Science topics: Cell BiologyMethods
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Questions related to Methods
Enrichment of CD4 TCells for tissue culture?
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Does anyone have advice on enrichment of CD4 T-Cells from blood of humans/mouse for propogation in tissue culture? A lab member is using the CD3/CD16 bead method for enrichment, but has poor results so far.
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There are a few other possiblities: Commercial, there is a Miltenyi T cell expansion kit. Also possible is to expand them via activation with platebound CD3/soluble CD28 and IL-2 or instead of CD3/CD28 you can use irradiated (or mitomycin c treated) APCs. With CD3/CD28 you will have more cell death somehow. Hope this helps a bit and leads in the right direction. Axel
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Hello,
I amplified a cDNA into pOTB7. I sequenced the plasmid and I got 99% similarity to my gene of interest.
I then RE digested this plasmid and RE digested pcI-neo vector and did ligation, transformation and sequencing.
Now in the sequence I get over 10 mutations for each primer I used. I am not sure what to do next because I have to use this plasmid for transfections downstream.
Can anybody explain why the mutations come at this stage and what I can do about it?
Thanks.
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Hi Neeraja,
I would like to know if u do an agarose gel purification after the RE digestion. With my experience what I noticed is that prolonged exposure to UV light during the time of excising the band results in so many mutations. I think u can have a check over it . And I wud like to know if u do a double digestion to ur plasmid and vector...
Regards,
Aishwarya Sriraman
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Hi, Ethididum bromide spilled on my lab coat while working. Fw drops of ethidium bromide was spotted on my trouser. I was not aware of that one for an hour. After one hour I went back my home and i washed for 5 minutes with water and soap. I am bit scared of this one. Does any one have experience like this. How it work.
Thanks
Madhu
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I won't eat margarine anymore :)
beta-cyclodextrin
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Hi, has anyone used beta-cyclodextrin to dissolve drug compound? Can anyone advise me on the best way to dissolve cyclodextrin? What is its maximum solubility? Am not familiar with it. Thanks for any advise
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Try dissolving in distilled water or double distilled water.
Vibrio question
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Hi, I know that TCBS media can be used to isolate pathogenic strains/species of Vibrio, but is there a selective media for non-pathogenic Vibrio. What would be the quickest was to go about identifying Vibrio. Thanks!
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Hi, I am now working for detection of Vibrio parahaemolyticus in raw vegetable and now looking for some journal about Vibrio parahaemolyticus specific in raw vegetable. Could anyone please help me a hand or any advice for me on that?
Designing primers for DGGE
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Hi, I'm trying to design a DGGE primer set for a gene found in a group of bacteria. However, there is no really good conservered area in the gene sequences between the different species. (Ex. there's an area where 9 bases match up, but then next bp does not match, and the next 5 does). What will be the best way to design primers. Thanks!
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Hi, i am new to the DGGE primer set, i am going to research on the microbiome analysis through DGGE, but i want to ask is there any DGGE primer set for universal bacteria? and is there any DGGE primer set for sepcial type of bacteria( such as Lactobacillus)? thanks!
Isolating bacteria from roots
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Hi, I'm looking for a protocol/method for isolating bacteria from roots. The plants that I'm collecting the roots from are Spartina and Juncus grasses.
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Check the following files: http://www.filefactory.com/file/aga623g/n/D_56_pdf and http://www.filefactory.com/file/aga6231/n/608_pdf click on "Download for free with FileFactory Basic " and enter the code and start downloading
Ligation Failure, highly frustrating!
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Dear all, thanks for reading. I hope you can shed some insight onto my problem::: I am trying to insert a 44bp insert into a 7.63KB commercial plasmid. I am cutting ~ 2.0ug with two NEB REs (Both 100% in Buffer 4 @37C 1hr) I then heat inactivate and run on 1% agarose gel, extracting the band with qiagen qiaquick column (horrible yields but much quick than phenol cholorform and no chance of loosing the pellet) Both enzymes are working, I have run these seperately to check. I am not dephosphorylating the plasmid. I am getting no/very little background colonies transforming with cut plasmid and no insert so I am sure I not need to dephosphorylate. The oligos are IDT standard desalted custom DNA oligos. I am worried about the purification but I know this standard works for other groups. I anneal the 2ug of each Oligo according to the commercial protocol, in 50uls annealing buffer I know that they are annealing (I have checked this on a gel) My insert is added in concentrations according to the protocol which is 50ng plasmid to 4ng insert. I have also tried ligating: 50ng: 8ng 50ng: 80ng 80ng: 160ng 80ng: 320ng All to no avail. I get no transformations on my plasmid+insert ligations, only my controls work. The ligase works (I have tested it each time with singly cut plasmid I am getting about 100 colonies on single digests) The cells are transforming very efficiently with control plasmids (3000 colonies with 0.1ng Puc1 DNA) I am getting very low background (1 colony per plate) with no insert so the RE digests are fine. Because I am doing miRNA target validation I will be trying to do this with a high number of inserts and continously for a fair few months (therefore keeping the costs down is a little important) If you have any ideas then please let me know.
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Trying to digest such short DNA fragments is not that easy. Some enzymes need a number of bases between the restriction site and the end of the fragment for the digestion to be efficient. There is a table with minimal requirements for various enzymes in NEB´s catalogue. What I usually do, is to order the oligos in such a way that the corresponding cohesive ends are formed (e.g. the ds DNA is not blunt). Such an insert can be of course phosphorylated in vitro, but this, again, is cumbersome. The best thing is to order each oligo with a 5´phosphorylation (most companies can deliver such oligos) and voila! In general the cloning is very straightforward. I have done this a number of times and it always worked. I hope this helps. Cheers
Nitrosomonas, Nitrobacter & Nitrospira
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Hi Everybody, I was wondering if anybody in Australia is currently working with Nitrosomonas spp, Nitrobacter spp or Nitrospira sp at the moment? I need to source an aliquot for a starter culture... Kind regards
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Hi! I have the same question for Europe...did you find somenthing?
Culture Media & technique
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Hi everybody, I'd firstly like to say hello to everybody - i'm new to the forums and am glad to have found you all. I am a Microbiologist in Melbourne, Australia and am currently working on a project for treatment of waste water and bioremediation principles. I'm currently working at culturing nitrifying bacteria - Nitrosomonas spp, Nitrobacter spp & Nitrospira spp I was wondering if anybody would have any sources that describe an appropriate culture media? being chemoautotrophs you can't just throw them on some nutrient media and hope for the best. I have come across two formulas: Formula 1 (NH4)2SO4 KNO2 CaCl2.2H2O MgSO4.7H2O Bromothymol Blue K2HPO4 (0.2M) KH2PO4 (0.2M) Chelated Iron FeSO4.7H2O EDTA disodium with the following trace elements NaMoO4.2H2O MnCl2 CoCl2.6H2O ZnSO4.7H2O CuSO4.5H2O Formula 2 (NH4)2SO4 K2HPO4 NaH2PO4 MgSO4.7H2O MnSO4.4H2O CaCO3 Some similarities but one more complex than the other...any thoughts or recommendations? do you have something that works for you? I am making 1,000litres of this media and want to grow up a starter culture in that 1,000L IBC (It's a fully sealable plastic shipping 'tank' which is wrapped on the outside with a steel frame for strength and support and comes on a steel pallet for shipping) Any advice would be appreciated... Kind regards and thanks for your time
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The DSMZ has a good collection of recipes. http://www.dsmz.de/microorganisms/html/bacteria.genus/nitrobacter.html - visit this and choose one of the strains, there will be links regarding culture media. I am also looking for a mineral medium for experiments on Nitrobacter in August, but I cannot use the ones with organic compounds, so if you find a mineral medium at the DSMZ and it works for you, tell me which one you used.
DIG Northern Starter Kit from Roche
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Dear all, i have a problem with northern blotting using the northern starter kit from roche. I have several probes, against my target mRNA (sense and antisense), and against actin and GAPDH as controls. I use to see very strong bands at the height of the rrna with all of the probes (except the one sense negative control) and do not know how to get rid of them. The technical support of Roche does not have any advice. I already tried lowering of probe concentration and prolongation of prehybridisation time, but this did not improve the result. The hybridisation temperature i use is 68°C. I also see specific bands for my probes, but the rrna bands are very disturbing. I know i could isolate only mRNA, but shouldn't i also work with total RNA? It would be nice if somebody could give me an advice, Kind regards, melanie Thelen
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Hi, The first think I can think of is that maybe your probe is not specific enough for your mRNA... did you check it well that you have no other sequence matches? If this is the case, you might think of designing an antisense competitor (unlabeled probe) for your rRNA's to decrease the background.
Swarming, beta-hemolytic bacteria, isolated from nostril.
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Hello everyone, I'm a newbie and wanted to ask a question that has been bugging my professor and me for quite a while. For Microbiology class, us students were supposed to isolate Staph. aureus and Staph. epidermis from our own nostrils with a sterile swab, and streak-plate them. I performed the correct procedure according to protocol, incubated it for the same time as everyone else, and sterilized everything that needed to be sterilized. One exception: I had some slight blood on the swab as I had recently had a nosebleed. However, my results turned out completely different from everyone else's. We plated the nasal flora onto TSA and sheep blood agar plates. After 48h of incubation, I discovered that my cultures resembled nothing like Staph. aureus or Staph. epidermis. On the sheep blood agar, there were colonies growing in the areas I streaked. They weren't very distinct colonies and appeared to be capsules, which were probably those of Strep. pneumoniae. What stumped everyone was the fact that the ENTIRE PLATE had turned brown, even in the areas I didn't streak. There was evidence of swarming with a rather undulate pattern away from the capsule bacteria, that just looked like a slightly rough, thin film, which isn't quite consistent with the Proteus genus of bacteria. On the TSA plate, the agar had turned from yellow to yellow-brown. The capsules were present, but some had a slightly pink tinge, while others were a translucent yellow/white. I isolated part of a colony and performed another streak plate on TSA, which showed more indication of swarming and agar-darkening. We were all very curious, so I performed a smear and a Gram stain. It was definitely Gram negative, and extremely small. Small enough that at 100x under oil immersion we couldn't tell if it was cocci, rod-shaped, or diplococci. I haven't performed a catalase test yet, and I'm currently trying to gather enough information to try plating it on different antibiotics. This really isn't related to my schoolwork per se as I was allowed to borrow some of my lap partner's Staph epi for further labs, but my prof and I are just really, really curious. If anyone could lend us a hand in identifying this bacteria species, we'd greatly appreciate it.
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Hi! If you have already performed microscopy you should more or less know if it is pure culture you have, or heterogeneous. If it is a pure culture for sure there are many ways to identify them like substrate utilization tests, capsule and spore staining and tests for different enzymes such as catalase. But these are quite labor intensive. Therefore, if you have sequencing facilities I would offer you to amplify the 16S gene with some general primers like GM3-GM4 or for even longer sequences 27f-1392r and use this product for sequencing. Then you can BLAST the sequence and see which bacterium it identifies with. Even if you don't have a sequencer of your own there are many companies who do it, and it doesn't cost that much.
protein disappeared from the gel.
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Hello everybody I am new in my current lab.I realized that all stock solutions-tris solutions for gel,commasie blue and %20 SDS- stay for a long time on the benches.However, I checked the pH.There is no problem.We prepared fresh running buffer and run the gel using samples with loading buffer which is stored in -20.Also we loaded markers.Nothing seems on the gel.we prepared new sample with SDS loading buffer.same things happend.Also I refreshed all Tris solution.Again the same thing... In addition,there si a problem with the appearance of the running gel. the stain should run as a particular band.however,there are lower and upper borders.and the stain run dispersed between two borders. what is the problem?
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first of all, if your protein is not on your gel, it is probably too DILUTED to get a reading. Secondly, you need to make sure that your protein DOES NOT run out of your gel. Third, if you are seeing smearing, you are running your GEL to at a VERY FAST voltage. LAST, make sure that your component in your gel and the running buffer is similar. If your gel's PH differ, it may only affect the migration because of protonation/deprotonation of the amino acids on your protein.
How to make a BAC library?
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Hi, I am wondering if anyone have any experience in making a BAC library? My aim is to construct a BAC library for a fish species, but I have never done this before and I don't know which protocol to use or where to begin. I have tried to search reserach papers to find a good protocol, but they are all so short and different. If anyone have any experience in purchasing such a library, I would also appreciate feedback, especially regarding approximate price and delivery time. All answers will be highly appreciated.
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I do an up on this question as I am also looking for a bac construct of human DNA not available in main companies selling this kind of material (but available on pubmed)
Nuclear protein extration for Co-IP
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Hi, Does anyone of you have a good protocol for extracting nuclear proteins? Im going to study protein interactions/complexes by co-immunoprecipitation of nuclear proteins. Thanks.
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Hello.. To extract the nucleus protein.. You have to select the lysis buffer. Because, Nucleus membrane doesn't rupture well. So, Many people use a RIPA buffer. And you have to physical method. You had better use a sonicator. Sonication of protein does't effect of protein interaction. When you break the nucleus membrane, you may chek the cell morphology by microscope. May be... It would be scattered. And then, Centrifuge the proteins. You will be success! In case of me, I use a RIPA buffer. The components of RIPA buffer : 50mM Tris HCL pH8.0 150mM Nacl 0.5% sodium deoxycholate 0.1% SDS 1% NP40
VADLO search engine
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Most definitely look up this search engine. http://vadlo.com You will find millions of protocols, databases, online tools and software. Also specific search category for powerpoints. And all of it cleanly categorized, so that you don't get journal articles when looking for a simple bench protocol!! Don't forget to check the daily research cartoons, and send them to your friends. They will appreciate it. http://www.vadlo.com/Cartoons/Life_in_Research_Cartoon_1111.html Maya (BTW, I tried sending this wonderful resource to the group, and the moderator apparently decided that the members of this group don't need it. You decide:)
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i prefer research gate
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Hi,
I have recently been a bit confused by the T4 RNA ligases... I know they are being used for 5' and 3' RACE, and also for RNA end labeling... Does anyone know if I can also use them to tag total RNA at the 5' or 3' end with some RNA oligos, perhaps about 20mers or so? would I have to do some modification on the RNA, like removing phosphate caps?
Thanks!
Basak
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Yes there is a way. I just remember you have to put a 5'Phos on your primer you are wanting to ligate. Look up some info on FLAC (full length amplification of cDNA) or SPL (single primer ligation) and that should point you in the right direction. Here is a link to one
mRNA extraction method
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Does anyone knows a method for the extraction of mRNA without using the extremely expensive magnetic beads? Thanks Guido
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Seems good, I will try it Many thanks Guido
How to sterilize cover glasses?
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Hi, I am wondering how you sterilize cover glasses? In my old lab we used to wash them really good and then rinse them with 70% ethanol and place them under UV for 30 min. Do you know a better alternate?
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For microscopy? We have always dipped in 70% ethanol and then flamed. This works fine and are sterile enough, considering we also grow cells on them in tissue culture.
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Hi,
Getting hints to solve one's problems is one of the main things on ResGATE. This indeed is fine but normally the community is not informed whether or not the information helped to resolve the problems and useful information is lost.
Therefore it would be a nice idea if we would fill this area to post our problems AND how we solved it. This would result in a very good and helpful (data) collection just like a troubleshooting guide
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Hi,
Well, it's not really like "troubleshooting", but last few weeks I was absolutely unable to get any RNA from my bacteria all of a sudden, without no reason... then Nasar told me just to relax and take some time off and have a fun and do it again.
Well, I tried, I just gave it a break for a week and did other things, went home early and didn't worry much.. then I tried, the same solutions, same cells, same protocol... It didn't work for weeks, and now it works again. I didn't change anything else, but perhaps it was a good idea:)
problem in cloning experiment
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dear my colleagues i have a problem in cloning experiment using PT7Blue T vector my insert is around 1500bp and when i made ligation using the previously mentioned vector . i got blue and whit colonies as usual but the problem is neither the white nor the blue colonies contain the insert after cutting with hindIII and EcoRI restriction enzymes , i only get very small insert aroud 80 bp. i do not where is my insert and what is the problem please if any one facing the same problem tell me how he can overcome it thank you so much
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Dear Rose, I've never used the PT7Blue T vector, but i had some problem too with other vectors also of the blue series. Maybe when you cut the insert from the plasmid you are doing a too strong restriction that is going to broke your insert in fragments. Did you check the entire plasmid+insert construct weight in agarose gel? Does it correspond to your plasmid+insert weight desired? Are you sure that your insert haven't any cut site for HindIII and EcoRI (or other sequence of cutting) in the sequence? 'cause maybe you cutted the insert with the same buffer for both endonucleasis, and maybe they had some "star activities". But if you wanna be sure, try to sequence the plasmid+insert construct, it's better. Andrea
i want to buy gel analysis software
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dear colleages i want to buy gel analysis software suitable for making densitometric analysis of a gel photo giving data like molecular weight of each band, its relative mobility ,.....etc please if any one can tell me what is the most suitable software ( i need the name of the software and the company name) thank you so much for your kind help
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What instrument are you using to collect the initial image? Shouldn't that company sell a suitable software? I use the biorad quantity one software, or NIH imageQuant. There are free solutions available via add-ons for "ImageJ".
problems with DNase treatment- using TURBO DNA-free™
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Hello All, I am trying to treat my total RNA before microarray. Using TURBO DNA-free™ but does not seem to work. I would appreciate any help and comments like which is step is crucial, how it does not treat anything? Here some trials I made: 1. DNA before treatment 224 ng/ ul 260/ 280 = 1.87 DNA after treatment 146 ng/ ul 260/ 280 = 1.81 RNA before treatment 145 ng/ ul RNA after treatment 118 ng/ ul Used 1 ul DNase in this one, DNA is still there with high quality! RNA is degraded too! 2. DNA before treatment 325 ng/ ul 260/ 280 = 1.95 DNA after treatment 253 ng/ ul 260/ 280 = 1.92 RNA before treatment 263 ng/ ul RNA after treatment 184 ng/ ul Used 3 ul DNase in this one, DNA is still there with high quality! RNA is degraded more then DNA Thanks
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This depends strongly on the protocol and the type of tissue. Normally I do a DNase treatment at the end of RNA extraction with an additional phenol/chloroform extraction
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Hi everybody,
We want to replace our old imaging system for qualitative DNA/Agarose Gels (working on Win 3.11!!!) and I wander if it would be useful to switch from EtBr to CybrGreen.
How does CybrGreen perform?
What hardware/system do you use (camera, luminescence, ...)?
Is its sensitivity comparable to EtBr?
Is it less cancerogenous (working with EtBr at our lab is a pain in ...) so that we could circumvent buffer clean-up and so on ... like we have to do for EtBr?
Is CybrGreen more expensive?
Thx
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Hi!
First of all I have to say that SYBR Green is definitely not less toxic than EtBr, so you have to be as careful... (although the manufacturers claim that it is less carcinogenic) It is also free of the complex waste disposal issues of EtBr... It is the best to have separate places for staining the gels afterwards and not contaminating the electrophoresis tanks with it. We have a separate room for gel staining and taking pics, and in that room we also have separate contaminated and non-contaminated areas, and there are snipers waiting around the corners waiting to shoot people who disobey the rules:)))
There are several different types of SYBR Green dyes, SYBRGreen I is specific for dsDNA and I think almost ten times more sensitive than EtBr. SYBRGreen II is more selective for ssDNA and RNA, and although it also binds dsDNA, it has a way higher quantum yield with ss molecules, therefore it is really good for RNA gels, also you can detect much less quantities.
You do need to use a different transilluminator for it, we have this Dark Reader transilluminator which uses visible light to excite, therefore you don't need no worry about uv. It is also very useful for cutting bands for exactly the same reason:))
Two things you need to watch out for is that it does not penetrate as fast as EtBr, therefore you need to stain 30 mins to an hour. And, you need to prepare the dilution in TE buffer with ph 8, that way it is stable at 4 degrees for some weeks. If you prepare it in water, it will disintegrate within a day and if the ph is not 8, it will not stain properly:))
And yes, it is a bit more expensive than EtBr but you also need less of it and it is more stable, therefore I would go for it:)) The results you get are especially way better with RNA and small amounts of PCR prods.
Hope this helps!
Happy weekend,
Basak
SDS PAGE smiling effect
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Hi everyone, i ran couple SDS PAGE in these few weeks.. and i am using Tricine SDS PAGE system, because one of my target was 4k. I am using 16% gel, and the formula is the following AB 50% 5ml Gel Buffer 5ml Glycerol 1.5g Add water to final volume 15ml APS 10% 50 microl TEMED 5microl Anode buffet = 1M tris, 0.225M HCL, pH 8.9 Cathode buffet = 1M tris, 1M tricine, 1% SDS, pH 8.25 Gel buffer = 3M tris, 1M HCL, 0.3 SDS, pH 8.45 All buffers are freshly made yesterday I ran at initial 30V, after the protein enter the stacking gel, i increase the voltage to 190V (i use constant V) i have totally 10 lanes, and the smiling effect was always occurred at around lane 1-2 and lane 9-10, and the lanes in between are ok. I also put the whole system in ice while i preforming the run. I have two questions.. 1) how to get rid of the smiling effect ? 2) Will the glycerol cause the smiling effect? For question 2, i asked because i did try to use the same buffer, but i didnt add the glycerol, therefore, i am wondering the smiling effect was caused by the glycerol... Can anyone help me ... thanks a lot .....
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Hi Danny, it seems the run @190V is to fast. I would sugest to decrease to 150V. How did you blot your protein? Yves
5' RACE: primers with a low melting temperature_troubleshooting
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I am using a SMART RACE cDNA Amplification Kit from Clontech in order to get cDNA from a plant roots (Datisca glomerata). I have been able to get few clones from cDNA amplified with primers that have high Tm values - as recommended by the protocol. Unfortunately, now I have to work with more troublesome sequences, where best primers that can be designed have a Tm value <64C. I was trying to follow the protocol without much success. Does anyone have some experience with this kit or some suggestions how to approach the problem? I am new to RACE, so I am not exactly sure how to troubleshoot RACE conditions. Thanks!
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If u want to do with total RNA n ur probable amplification product is less than 1 Kb thn u can use 5' RACE System of Invitrogen. I think for less than 1 Kb it is the best one n it always works.
Boyden Chamer Migration Assay
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I am using PC12 cells to study chemotactic migration. I am having a hard time finding a positive control. Does anyone have an idea for one or has used one?
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Why not use a typical line used in migration papers, something like PC3 or LNCap cells? You could also try a macrophage line (not raws) or one of the other primary lines known to be motile.
Northernblot
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Hi everybody!! I am having a very stupid problem but i cant get a logical cause. It is the first time i set up a northernblot and somehow is not working at all. I am working with RNA from S. coelicolor M145. The method i am using is northernblot by capilarity because on my lab we dont have any machine to blot. My specific problem is that i can not transfer the RNA from the agorose gel to the membrane. I already tried to let the blot longer, with more buffer, with more weight, but nothing is working. The RNA agarose gel is made with formaldehide. The buffer i use to blot is the SSC 10X (NaCl 1.5 M, and Sodium Citrate 0.15 M). Do you have any idea about what is happening?, Do you know if i have to make any other treatment to the RNA, or to the RNA agarose gel before blotting?......PLEASEE HELP!!!!....I will be really glad for your answer.
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Did you denature RNA before loading samples in agarose gel? Did you prepare the gel with a saline solution before blotting? wash with H20 (milliq!!!) twice and then NaOH 0.05N -0.1N 30 min in shaker, do not wash gel after NaOH...if i remember well... Buffer SSC10X is right, however soak gel in SSC20X before transfer. Even if I think that capilarity is not enough...you need a vacuum pump on the bottom of your membrane... only one last question, what's your agarose gel concentration?
Northernblot
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Hi everybody!! I am having a very stupid problem but i cant get a logical cause. It is the first time i set up a northernblot and somehow is not working at all. I am working with RNA from S. coelicolor M145. The method i am using is northernblot by capilarity because on my lab we dont have any machine to blot. My specific problem is that i can not transfer the RNA from the agorose gel to the membrane. I already tried to let the blot longer, with more buffer, with more weight, but nothing is working. The RNA agarose gel is made with formaldehide. The buffer i use to blot is the SSC 10X (NaCl 1.5 M, and Sodium Citrate 0.15 M). Do you have any idea about what is happening?, Do you know if i have to make any other treatment to the RNA, or to the RNA agarose gel before blotting?......PLEASEE HELP!!!!....I will be really glad for your answer.
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Did you denature RNA before loading samples in agarose gel? Did you prepare the gel with a saline solution before blotting? wash with H20 (milliq!!!) twice and then NaOH 0.05N -0.1N 30 min in shaker, do not wash gel after NaOH...if i remember well... Buffer SSC10X is right, however soak gel in SSC20X before transfer. Even if I think that capilarity is not enough...you need a vacuum pump on the bottom of your membrane... only one last question, what's your agarose gel concentration?
Bacteria and Paraformaldehyde
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Hey guys, I'm planing a research trip to collect some samples and my advisor just threw at me a new type of sample to collect. I'm making a homogenate of root material to plate onto media. My advisor is wanting me to take some of the homogenate and fix the cells with paraformaldehyde to perform some FISH studies. The problem that I'm forseeing is how long can the cells sit in paraformaldehyde before they are damaged? Doing the lit search and protocol search, I've only seen that they let the cells sit in paraformaldehyde for 24 hrs before filtering. Can the cells sit longer? Thanks!
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Hi, No, the cells cannot sit longer, otherwise you won't see anything. I think even 24 hrs is too long. You could look at http://www.arb-silva.de/ for some more protocols.
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Hey guys, one question...
I am trying to do an IP followed by elution for native gel run... But I have no idea how to elute the IP product out... definitely no boiling or SDS or low pH elution buffers... Anyone has any idea?
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I am doing sputum processing and when I finished from the haemocytometer count, I got total cells of 0.67, I then centrifuged the filtrate of the sample and then remove the supernatant leaving the cell pellet, I need to know how THEN I resuspend the cell pellet in PBS then adjust the cells/volume to give me 0.5 million for preparation of cytospin slides! Or How much PBS should I add on the pellete to adjust 0.5 million cells/ml? I get confused with math here!! Thanks for help
Hi I have just joined
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Dear all I am working as a post doc in University of Leeds, UK. Our lab is working on Purnergic signalling and how this ligand ion channel is modulated. I would like to have productive discussions with you. Thanks, Regars, Venketesh. S
How much PBS should I add to cell pellet to give me 0.5 million?
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I am doing sputum processing and when I finished from the haemocytometer count, I got total cells of 0.67, I then centrifuged the filtrate of the sample and then remove the supernatant leaving the cell pellet, I need to know how THEN I resuspend the cell pellet in PBS then adjust the cells/volume to give me 0.5 million for preparation of cytospin slides! Or How much PBS should I add on the pellete to adjust 0.5 million cells/ml? I get confused with math here!! Thanks for help
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Why dont you resuspend in PBS first, then count? That'd be the easiest way...
Rats and Mycoplasmosis..suggested treatment??
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Hello! I have come here in a desperate attempt to find solutions to treating mycoplasma infections in rats. I am hoping someone here may have some suggestions or advice! I am a caretaker of four male rats ranging between the ages of 2 years to 9 months who have been suffering with myco flare-ups off and on throughout their lives. I have treated them with weeks of baytril/doxy combo, as well as zithromax alone and along with the baytril/doxy combo at the first signs of sneezing and wheezing. My two older boys have significant lung damage despite treatment . My two younger boys I believe are doing well, but I have been trying to find solutions to help prevent flare-ups or stop/reverse any damage that may be occurring even without symptoms. I have currently taken a difference approach and have been trying different supplements such as Co-enzyme Q-10, Alpha Lipoic Acid, salmon oil and have considered starting my most cronic boys on N-Acetyl L-Cysteine (does anyone know a safe dosage for N-Acetyl L-Cysteine for rats? They are 670 grams and 560 grams). I am hoping someone could give some advice or suggestions on how to better treat this awful mycoplasma, as I have become so desperate. Thank you VERY much for taking the time to read this, and thank you for any replies that follow! Your feedback is very much appreciated and I could not thank you enough!!! - Debbie
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Hey, thank you for replying! It's been a while since I've logged on and unfortunately I missed the reply. Sorry for the 11 year delay!
question about double digestions with fermentas restriction enzymes
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some times when i want to build a clone with two enzymes to cut DNA, fermentas recommends a double digesting system i which some enzymes may require a 2/4 folds of adding. In this situation, shoud i do a double digest or two single digests? thank you.
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Its upto you! I also use fermentas restriction enzymes. What I do is that I cut the fragment and vector with two single digests before ligation while in order to confirm the insertion of my fragment into vector, i do double digest using their recommended protocol. Just follow their protocol, if you are afraid of increased buffer concentration in the solution then digest fragments for 2-3 hours and do single digestion for enzymes showing star activity e.g. BamH1. I don't like doing overnight digestions unless the enzyme i have is very expensive. I believe in "non-stop" cloning as to minimize the possible mutation with the nucleic acids i work with.
problems with DNase1 footprinting
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Has anyone ever seen DNA NOT go into a denaturing 7.5M urea sequencing gel? I am losing the bromophenol blue band altogether and the DNA fails to migrate more than 1/10 of a mm into the gel. I don't think it is a salt problem, and I'm running in 1X TBE which is the same as the gel. The volts start off ok and climb so power supply is good. Anyone ever seen such a thing and how do you fix it? thanks.
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Perhaps the fragments are too big and the gel is too concentrated? Or you are loading too much?
!DISCREPENCIES BETWEEN LAB TECHS!
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I've been trying to figure out for a while now why my co-worker and I get such different counts when we plate (we use PetriFilm). My counts are always quite a bit higher. When we plate side-by-side, we do everything the same. Sanitize the counter, wash our hands, and start plating. The peculiar thing is when we do the American Proficiency testing every quarter. In the past I've had counts way higher than hers, but mine seem to be the average in comparison to all other facilities participating in the test. When she does it by herself, she tends to score 50-75% whereas I always score 100%. Is it possible she is somehow actually KILLING bacteria when she plates?? Some products that we test require us to aseptically cut off pieces of raw meat and stomach it with Peptone. The aseptic technique we use is scissors and forceps stored in alcohol. I always shake off excess alcohol prior to cutting afraid of killing something on my sample. In school I learned that alcohol sanitizes as it is drying and once it is dry, it is no longer effective. Therefore, by removing the excess, it allows the utensils to dry a bit before coming into contact with the meat. Does this sound like a possibility as I don't believe she does that prior to cutting?? I'm confused! If our test results from American Proficiency Institute were flip-flopped then I'd guess I wasn't being sanitary enough so that's what throws me off! I guess that still wouldn't explain the fact that even when we don't use utensils (such as with the proficiency tests), I still get higher counts. Any ideas??? I'm stumped!
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You might try doing about 10 plates from a single sample. For the first five, do your usual method of shaking the alcohol off of your tools. For the second five, leave the tools wetted with alcohol. Incubate, count and compare the plates. If your hypothesis is correct, you should see counts on the wet-tools plates averaging about 50%-75% of the dry-tools plates.
Cell line authentication
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I am looking at performing some cell line authentication. I am wanting to be able to confirm no cross-contamination between murine, chinese hamster and human lines as well as between different human and chinese hamster lines. Has anyone any experience of either using any of the commercially availible kits or performing their own system for authentication? many thanks Rainy
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HI Rainy, iQ Biosciences offers this service. Have a look at this webpage for very detailed information (below). A lot of journals are starting to require this for publication and even funding societies, etc. http://www.iqbiosciences.com/bioservices/testing-services/cell-line-authentication Best Regards, Omar
Question
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Hi,
I have to perform an experiment to see if my bacteria have strong restriction enzymes that are degrading the plasmids I am giving them... for that we had the idea to extract the bacterial proteins and incubate them with the plasmid, to see if it still stays as it was or if it's digested... the problem is that most extraction methods I have include cell lysis with urea and detergents, as well as grinding or sonication and I really don't think that the enzymes will still stay intact after such a procedure.. On the other hand I guess the DNAases will also still be there.
Any ideas to extract potentially active proteins from bacteria? Or is it just not feasible?
Thanks!
b.
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Umh, I am not really an expert in protein extraction, but I remember that we once purified an overexpressed protein from bacteria, and cell lysis was done by sonification. Something like 10 minutes for 30 g of bacteria. After that several chromatography steps followed and in the end we had a nicely working, native protein. So I think you can try that. Another method you could try is lysis with a microfluidizer, if you have one in your institute. The problem I see with using the crude lysate for incubation with your plasmid is that you probably have tons of genomic DNA in there... So you might need to get rid of that first.
I hope that helps a bit.
-Jan
Bacteria and roots
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Hey guys, I'm getting ready to start a new project where I'm isolating bacteria from roots and I have a question. My current plan is to grind the roots up and perform a serial dilution on a set of roots. My question is to what dilution do I need to carry each set out to? (Ex. 10-5, 10-6,etc, etc.) Thanks!
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Hi, I would start with 10-12 or so, and afterwards you can see in which tube no bacteria are growing, which would be the average number of the cells.
Tissue preparation for electron microscopy
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I am performing the perfusion-fixations of several rat brains, which will then be further processed by another lab for electron microscopy. My question is: If I only do the initial paraformaldehyde/acrolein fix, how long can the tissues be stored in fixative prior to secondary fix and embedding at the other lab?
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We fix the tissue in paraformaldehyde for 1 hour and then neutralize it with buffer immediately. then we store the tissue in 4 degree Celsius. In our case further processing take place within a week. EM image appears good and clear. We are studying intestine and colon sections from mice.
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hi everyone. I am very new in this field. If anyone can help me out, I would be so grateful:
I am working with chicken ileum to detect and quantify bacteria.
1. Anyone have a protocol for real-time PCR using 16S rDNA?
2. Can give me a site where i can find 16S rDNA primers for bacteria such as Lactobacilli, Bifidobacteria, E.coli?
3. I am trying to design some primers by using the NCBI databases, there are so many strains of lets say Bacillus cereus...how do you know which one to choose?
Thank you for replying
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Question 1: see for example a paper from our lab (shameless self-promotion!):
Microbial Prevalence, Diversity and Abundance in Amniotic Fluid During Preterm Labor: A Molecular and Culture-Based Investigation, PLOS ONE 2008, 3:8 (e3056).
Question 2: harder to answer quickly, but I'd suggest doing literature searches. There are many of such PCRs developed, and the answer would depend on whether you want to detect a specific species or a whole genus.
Question 3. In general, you would need to compare all the 16S genes of the species/genus you want to develop a PCR for, and pick specific primers that will work on all of these. I use the program ARB for that, but that's a steep learning curve, and then test my primers for specificity in the rdp database at http://rdp.cme.msu.edu/ (probe match function). But again, doing a literature search would be the best approach, especially if you are interested in detecting groups that are well characterized. Other groups probably already have published specific PCRs for that.
Good luck!
Histone immunoprecipitation
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Hi, I need help with histone immunoprecipitation. I have never worked with histones before. I want to study if my protein is associated with any of the 4 core histones and 2 linker histones. What kind of lysis buffer, sonication settings etc. do you use? I work with mammalian cell lines. Thanks!!
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same no difference..
Exonucleases to clean-up gDNA
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Has anyone ever tried cleaning up gDNA contamination in a maxi-prep with exonucleases. Im thinking of trying it but dont know what on to use Thanks
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if your sample is available in extra amount u should try. u will apprantely see the results
TCBS Agar
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I was preparing for a sampling trip this week and something came up and the trip has to be postponed until the last of this month or the first of May. However, I've already made lots of TCBS Agar plates, and I was wondering if the plates would still be usable/good for the next trip? Thanks
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Hy, It still be usfull untill the next month in case you put it in a clean refrigator (not contaminated with fungus) at 4°C. Look at each plate before use if it didn't contain any contaminat use it. Best
Question
2 answers
Hello,
I am trying to dissolve glycogen for an enzyme assay.
it dissolves in hot water but is not clear, it is a cloudy solution.
I am wondering if glycogen solutions remain cloudy? If not, how do I dissolve it?
Thanks.
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you can dissolve glycogen in isopropanol (5-20mg/ml). and from this stock start with your enzyme assay.
Best..
How to check antibodies for viruses?
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We want to inject some of our animals with an antibody of which we have a clue that it will raise the blood pressure. we got a comment from the animal house that we had to take extra safety messurements, which is a drag cause they have to go nto a special room and we have to the the blood pressure over there in a flowhood. The reason is that it was suggested that the antibody might contain a virus. Abcam cannot rule out that there aren't any in there, so is there a way I can prove this??
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PCR for traces od viral RNA/DNA? Or you could try to purify those antibodies with Protein A - I doubt viruses bind to that medium.
growth of vibrio in TCBS agar
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hi..... i ve been growing vibrio for the past two months on TCBS agar. Previously it was changing the colour from blue to yellow. But from the past 2 subculturings, the colour of the medium is not changing but the colonies are still growing... Can anyone tell me what could possibly the reason....
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Hi! First of all, for PCR I would just get a tiny little bit of cells on a clean toothpick, throw it into 10 ul of PCR water and use 1 ul of that as template for PCR... no need for extraction. As for the sequencing, I would give you the protocol, but it only makes sense if you have a Sanger sequencer or a friend that has one:))\ Did you try inoculating the culture new on new media, or microscopy?
SDS-PAGE: loss of current
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I've recently switched to using an old vertical gel apparatus, which has larger gels - about 15cm. Running 2 8% SDS-PAGE gels at a constant 60mA, the voltage increases after an hour to 300V (The max our powerpack allows) and then the current starts dropping, eventually hitting about 9mA after 2 hours. I run the gels that long because I'm trying to separate 200-500kDa proteins. The resistance is obviously increasing which is why I get a drop in current. I thought this may have been due to overheating of the gel, but got the same effect after running it in a cold room (4degC). I also changed the buffer after about 2 hours, which raised the current to about 20mA, but my proteins went nowhere. I'm not sure why I'm getting such an increase and wonder whether I should try a different gel and buffer system. Can anyone help?
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First, have you checked the integrity of the wires in the system to be sure there is nothing unusual? Second, is your gel completely submersed in the buffer (inside the frame and out)? Lastly, is the concentration and pH of your running buffer correct?
Viral cell interaction
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it is very important to us to know the mode of interaction between virus and cell.
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A little more information may be helpful. Are you looking at virus binding to the cellular receptor? Or are you just meaning the whole scope of virus entry to the cell, replication etc. Which virus?
Has anyone had issues with guanidine thiocyanate toxicity??
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We have recently had problems in our lab with fumes making people ill. We have very few bad chemicals around, but I've recently started wondering if our PCR cleanup kit may be to blame - we use a 96 well kit and do up to 20 plates per day, so we are using more than the usual amount of this reagent. The MSDS says that guanidine thiocyanate may be harmful if inhaled and can cause respiratory sensitization. These are the symptoms we have experienced: burning eyes, burning lungs, dizziness, shakiness, headaches. We are extremely sensitive now, and even 30 seconds in the lab space can cause symptoms that last for several days. Others, such as consultants and our next door neighbors, can come in and have no problem, aside from possibly a bitter taste on the tongue or a scratchy throat. Has anyone ever experienced something like this? We have had to abandon the lab, four weeks ago now, and despite opening the windows, the problem seems to be getting worse rather than better. Any suggestions would be extremely welcome. Thanks!
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Guanidine thiocyanate is used for cell lysis and as such is a very effective combination of chaotropic components. This means when inhaled it will also do a similar job in your lungs. The guanidine might even pave the way for better resorption of thiocyanate. Thiocyanate can interere with the thyroid iodine resorption and also was used as an agent to fight hypertension. If you are spinning material in a centrifuge aerosols can build up quite easily and long-time exposure might lead to an ongoing sensitization of the contacted tissue (your lungs). Being only a biochemist it is difficult for me to give a "remote diagnosis". But for me it sounds like you and your colleagues might be exposed to lower doses over longer periods. Therefore avoid inhalation or direct skin contact and set your centrifuge into a chemical fume hood. Rule out other sources of aerosol building in your lab that could contribute to the thiocyanate to arrive at your lungs. Symptoms look like a mix of increasing sensitization and local damage of the lungs and maybe also the unwanted side reaction caused by the bioactive thiocyanate. Contact your local safety department and maybe also let a doctors have inspect your lungs to avoid longstanding lung edema and the like.
Protein level comparison method
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Dear all, I've heard WB is a qualitative method rather than a quantitative method. How if i only want to compare the protein level between samples, can i still give conclusion simply by direct comparison? My second enquiry is whether immunostaining also serve the same purpose to compare protein level between tissues or cells? Lastly, if immunostaining have such a application, which one is better in the sense to compare protein level, WB or immunostaining? Thanks a lot!!
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>my recommendation is WB with chemiluminescent detection and a >digital imager (eg Bio-Rad). I have done this during several years. It is tricky though, specially when using short-lived chemiluminiscent substrates. Since it is less sensitive than film, you generally need several minutes exposure. If the timing is wrong, then it is very difficult to obtain good results upon a second exposure. At present, we are using in my Department LiCor's Odyssey InfraRed detection system which is pretty cool: large dynamic range and very high stability of the labeling. You can go back and scan again your blot months later, provided you kept the membrane protected from the light. The equipment is quite expensive though, I guess arround 100 k$ but antibodies are quite inexpensive, and solutions can be often re-used 2-3 times. As compared to peroxydase-ECL-film-developer method, we have calculated a 3-years ROI. Furthermore, since you can analyze at two different wavelengths, you can do a sort of multiplexing (e.g. normalization using an internal control). There are also several very good primary antibodies already tagged with the IR-Dyes which allow a fast analysis without the need of secondary antibodies. Streptavidin-based labeling works fine as well.
Failure in cell detachment after trypsinization
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Dear all, I am routinely using trypsin to harvest my cells, but it happens that a few cell lines are unable to detached after trypsin incubation and tapping of the flasks, and situation didn't turn better even i prolonged the trypsin incubation time. Typically i will add 1ml of trypsin per 25cm2 flasks, and incubate them for at least 8 mins. It's problematic as i can only retrieve approximately less than 10% of cells from the flask, it minimized the amount of substances to be extracted out. Is there anyone having similar experience and kindly give me some advices? Thanks in advance
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I completely agree with Julia’s proposal of EDTA. For most of the cell lines I have been working with, I use to : -first wash briefly the cells with Ca++-Mg++-free phosphate-buffered saline (=PD,Versene), and -then incubate with a mixture of Trypsin + EDTA (commercially available). Trypsinization might be very deleterious for surface antigens, receptors, etc., so in many instances, you can also very easily DETACH YOUR CELLS WITHOUT ANY TRYPSIN, by just using Versene buffer + EDTA (a few minutes at 37°C or even at room T). This trypsin-free technique was especially effective to me when I worked with monocyte/macrophage primary cultures whose adherence appeared reinforced by trypsin !!!
How to do agitation?
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Dear all, Asi'm not from English speaking nation, i cant get the idea of agitation. Actually what should i do when told to agitate your sample, like solvent in eppendorf? thanks
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"Agitate" just means to mix, usually by shaking, rocking, or vortexing, depending on the sample. There are shakers, rockers, and vortexers available for such purposes at sites like the following: http://www.southwestscience.com/
Cell culture:RBL-1
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Hi I am currently using RBL-1 cell. It grows partly adherent and is mostly in suspension and we are culturing it in MEM. 1) Is it considered as a suspension cell line or semi-adherent cell line? We are currently monitoring the cell viability as the cell viability is low: <80%. 2)Should I be using the cells that adhere to the flask or cells that are in suspension? Currently, we did not typsinized the cells and is only using the cells in suspension. Thanks!
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Hi Nico Thanks for your response! Currently we leave the attached cells ( there is a smaller ratio of attached cells to suspended cells as they only grow partly adherent) in the flask and only pipette out the cells in suspension for our experiments. So you are suggesting that we should be splitting the attached and suspended cells in the same ratio during subculturing? In addition, should we be pipetting out both the attached and suspended cells or only the suspended cells for the experiments? Each have a different morphology, but we are using the cells to study the ion channels. Thanks
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Hi,
could I start a thread on an improved Laemmli buffer. Roti-Load is based on several papers showing that with phosphate buffer and lower temperature (65°C rather than 95°C) one gets less protein degradation. There is no need to boil Laemmli anyways; some transmembrane proteins can actually form aggregates.
What's your experience.
Harald
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Hi Nico,
thanks for looking into this. The 3 relevant papers that I found are:
(1) Cannon-Carlson, S. and Tang, J. (1997), Anal. Biochem. 246, 146-148.
(2) Kowit, J.D. and Maloney, J. (1982), Anal. Biochem. 123, 86-93.
(3) Rittenhouse, J. and Marcus, F. (1984), Anal. Biochem. 138, 442-448.
Carl Roth AG (Germany) sells Roti-Load R.
Proteins denature at 65°C, so I never understood why 95°C should be necessary.
Looking forward to hearing from you.
Cheers,
Harald
NF-kB binding activity in neutrophils and mononuclear cells
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I'm trying to stimulate human mononuclear cells and neutrophils with LPS to study the NF-kB binding activity by EMSA but I haven’t been successful yet. Can someone give me a protocol of this method? In my assay I isolate the cells from the blood, then I ressuspend them in Hank’s Balanced Saline Solution and let them stabilize for 30 min in a water bath at 37 ºC with soft agitation, and then I incubate the cells with LPS at different times. But I still can’t see an increase in the signal of EMSA. What can I be doing wrong?
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I have no experience with EMSA's from PBMC's - only cell lines. Perhaps you could try adding a little serum to the buffer mix, or even doing it in media- if the ficoll extraction etc takes a while the cells won't be too happy. Do you know how alive they are at that point?
adding egf to growing hk-2 cells
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Hi guys, does anyone have any information about culturing HK-2 cells? I'm trying to grow them... just wondering, do I need to add fresh EGF every few days to the media?? Thanks for any help, sara
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If you add serum in the cell culture media you do not need to add EGF, just change medium. I use DMEM + glutamine + antibiotic and they grow perfectly well. You also can use the KSFM medium from Invitrogen and then you have to add the supplements BPE and EGF, but first option is cheaper and it works.
frozen bone section trouble
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Does any one have any experience when cutitng frozen bone sections using a cryojane. Im having several problems... 1 when using the adhesive tape not all of the bone is transferring to the slide. 2. I need to cut cross sections of tibia but do not know of any molds that can embed the tibia cross sectionally and of anything I can mount the bone to so it can be cut on the cryostat. 3. I need a stain that's easy to use to look for the growth plate when cutting so I know how far I need to cut into the bone. Yeah lots of things I need to know and literature it difficult to find. If any one can provide any info that'd be great.
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Hi Julia, how are you doing? I heard you are having trouble and maybe I can help you out. There is a good article in Calcified Tissue International on sectioning iliac bone, which might come close to the tibia problem you are describing. Here is the link: http://www.springerlink.com/content/b28847m25r1m2844/fulltext.pdf?page=1 to your problems: 1) do you really need all the bone to go onto the slide? Sometimes it is sufficient to see the part of the section that is of interest. If yes, try to put the slide with a slightly higher temperature directly onto the bone, very slowly. Then bring the slide with the bone immediatley back to the cyrostat. 2) Refer to the article, it seems to have an applicable mold. If nothing else, the only good method is using methylacylate and cut the bone using a diamond blade. 3) I have used Toluidine blue to stain developing bone/growth plates. This was actually suggested by Fred Shapiro, who is a Hsitopathologist at Childrens in Boston, doing bone and growth plates in piglets histology and cutting on a regular base. You m,ight want to look him up in literature: Shapiro F, Childrens Hospital Boston, Harvard Med School. If you have some questions, he is a very nice guy and will answer your email. Christian
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Hi there,
I am doing an honours project using spores of B.subtilis and B.cereus.
I need to prepare a minimal media which does not stimulate germination but supports growth of the vegetative cells. I'm having a really hard time finding out what type of minimal media is suitable for these organisms and my requirements. Someone please help cause i am ready for pulling my hair out!
Thanks,
Alison
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Sounds difficult to me. You can keep vital cells viable.If they are finding conditions however where they can grow and mutiply this is typically also a signal for spores to germinate. Thats what they are up for. If the vital cells have been raised separately you could wash them in 0.9% NaCl (1 or 2 times) and store them for some shorter periods togther with spores in 0.9% NaCl.Not to long of course because they eventually will become hungry and depleted of food. Then if required you can add components of a full medium and observe what you want to look at...
Just an idea (I am not a bacillus expert however)
Measuring Ethidium Bromide without DNA
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Im doing a project regarding phytoremediation of EtBr on selected grasses. Im plan to use FT-IR and UV spectrophotometer. I need some help on my project. Ques1: I wonder if anyone know what wavelength to detect EtBr alone without any DNA? The EtBr will be planted in a plastic bag and soil with grass. Ques 2: Anyone got suggestion how to extract the EtBr from the soil or plant to measure it the EtBr% uptake by the plant. Thanks
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Hello, I know this is really a late response. How did your project go?
How to deal with so many false positive clones, Transfer linear DNA into E.coli competent cells...
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Hello, I have a problem in using Red recombination system to knockdown bacteria genes. I transfered linear DNA (long homologous arms) into E.coli competent cells by electroporation. But when I tested its sensitivity with kanamycin, there are so many false positive clones on the plate but no positive ones. How can I do? Thanks, Shao
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Check your homologous arms for false homology or repeat regions. Or simply shift the homolgous arms elsewhere. Long HR arms are good - but they increase the chance of false recombination, so try shorter ones too. I have gotten away with arms as short as 40 bp.
electroporation solution
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What kind of solution is the best to perform eletroporation pulse in in case of electroporation of chicken embryos in ovo?
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These might be useful: Voiculescu O, Papanayotou C, Stern CD. Spatially and temporally controlled electroporation of early chick embryos. Nat Protoc. 2008;3(3):419-26. Sauka-Spengler T, Barembaum M. Chapter 12 Gain- and Loss-of-Function Approaches in the Chick Embryo. Methods Cell Biol. 2008;87:237-56. Nakamura H, Katahira T, Sato T, Watanabe Y, Funahashi J. Gain- and loss-of-function in chick embryos by electroporation. Mech Dev. 2004 Sep;121(9):1137-43. Review. Krull CE. A primer on using in ovo electroporation to analyze gene function. Dev Dyn. 2004 Mar;229(3):433-9. Kos R, Tucker RP, Hall R, Duong TD, Erickson CA. Methods for introducing morpholinos into the chicken embryo. Dev Dyn. 2003 Mar;226(3):470-7. Tucker RP. Using Antisense Morpholino Oligos to Knockdown Gene Expression in the Chicken Embryo. Acta Histochemica et Cytochemica, 2002, vol. 35, no. 5, pp. 361-366
DsRed Expression problems
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Hi, I am have trouble with getting stable expression of DsRed in HEK cells. I am using the pStoplight Vector which expresses a LoxP flanked DsRed until it is cut out by Cre Recombinase, and then GFP is expressed instead. I have cloned this into the Flp-In kit from Invitrogen to generate a stable cell line using flp recombinase to integrate the vector into the genome. pStoplight info: http://www.ncbi.nlm.nih.gov/pubmed/11730010 Flp-In info: http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect&productID=K601001&messageType=catProductDetail&showAddButton=true It looks good for a while, but then, even though I have cells surviving the selection agent, after a week or so, I dont have any more red fluorescent cells! So, I have no idea whats happening. It seems to me that either: 1) the transient expression of flp recombinase is somehow acting on all the stoplight transfected cells, somehow removing the gene... unlikely. 2) DsRed expression is somehow being driven down by the cell? 3) DsRed is somehow toxic and kills the stable cells, but then why does anything survive!? Anyway, any suggestions or advise would be very much appreciated!
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The lamps don't really pick up red that well - and even doing confocal the red laser's are generally weaker. Make sure you have the right filters on etc to see it aswell.
10X Maleic Acid Buffer
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Hi! I wanted to prepare 10X Maleic Acid Buffer (traditional recipe: 116g Maleic Acid, 88g NaCl - adjust to 1L and adjust pH to 7,5 with solid NaOH). But I only have Maleic Acid Disodium salt... Can I use it? Maybe like this: 160g Maleic Acid Disodium (1 mol) 88g NaCl and then just pH eitheir way Can someone help me? Thanks, Diana
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i would use it in that way, as if the disodum was not there!! the NaCl ist for the Cl ions!! and at least when you will adjust the pH you also ad Na !! and did you ever calculated how much Na you have at the end, when you use NaOH? Finaly the best way to find out is to TRY! :) use 160g for 1M Malic Acid! and not 116 !!
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Dear colleagues,
can anyone provide a protocol or a few general hints for the usage of human transcriptome subtraction? Anyone who has direct experience with this method?
Thanks and regards
Sebastian
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Hi,
have a look at the thread 'RNA isolation from blood'
Problems with EMSA
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Hello, Is there anyone working with EMSA? I am new to this technique. My protein binds to DNA and gives a shifting band, but the problem is that I don't see a clear band, it has always a small tail. Is there anyway to avoid this? thanks.
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sum sum, to convert 0.5 ug/uL to nM you do it this way. 0.5ug/uL=0.5g/L; 85kDa is 85x10^3 g/mol; therefore, 0.5g/L*(1/85000 g/mol); now just convert it to nM. as far as your question regarding the protein binding to fragments, before you perform any assay, i recommend that your protein is extremely pure. If you have other DNA binding proteins, it may also bind to your DNA and give anomalous results. Secondly, EMSA is not a true equibrium binding assay, because the components are separated when run through a gel. And as you know, in an equilibrium assay, the rate of product and reactant production is the same. This cannot be achieved when you are separating the product and reaction. One must be extremely careful when calculating the binding constant through EMSA assay although it is possible. Secondly, it is important to do experiments with your protein closest to the binding constant because too less of protein concentration will require more protein to saturate your DNA but too high will completely saturate your DNA and binding constants cannot be calculated accurately (or close to accuracy). Third, make sure that your protein is in the correct binding buffer. Often after protein purification, your protein is not in the optimal binding condition; dialyze and then do experiments. Fourth, i recommend that you keep your DNA constant and increase the amount of protein to see if you get a change in "shifted band" intensity. I work with protein-DNA interaction too and I have to take in consideration of all the above. I hope this helps.
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Method links:
University of Nottingham, Pathology:
Western Blotting (Immunoblotting) commercial site
Mixture of Biochemistry methods, Genetic methods and molecular biology methods
Hahn lab, Fred Hutchinson Cancer research center
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Hi folks,
I'm Jean-Philippe, got a Ph. D. in management (strategic management). My work is focused on competitive advantage and I use multiple linear regressions and structural equation modeling. I'd like to go towards PLS methods because I work on more and more research questions requiring this method.
Anybody would have any advice, or some good paper to read?
Thank you.
Jeanfi
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Jeanfi ,
I would recommend you start with these two articles as well as the 2013 special issue from Long Range Planning on PLS
Hair, J. F., Ringle, C. M., & Sarstedt, M. (2011). PLS-SEM: Indeed a Silver Bullet. Journal of Marketing Theory & Practice, 19(2), 139–152.
Hair, J. F., Hult, G. T. M., Ringle, C. M., & Sarstedt, M. (2014). A Primer on Partial Least Squares Equation Modeling (1st ed., p. 307). Sage Publications.
actin severing / capping assays
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Does anyone have any experience looking at formation/degradation of actin filaments? If so, please share the assay you are using. I realise it's a rather technical experiment but seems doable based on the availability of commercial actin, polymerisation buffers etc.
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We use TIRF to assess actin polymerization and bundling with fluorescently tag actin
Hep G2
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Anyone working or worked with HepG2 cells? Need some tipps and tricks how to handle them. The main problem is that they are clumpy as hell, doesn´t matter how long i trypsinize them. They always form "mini livers" and are not distributed as a monolayer.
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HepG2 cells tend to grow in clusters, often forming aggregated 3D structures. Don't let your cells to over grow, they will form clusters than monolayer if you wait too long. Harvest them ~ 70% confluence with help of trypsin. wash cells with warm PBC/incomplete media and break pellet very gently to disperse cells into single cell suspension. I use 5ml syringe and 18 gauge needle for this. and it works fine for me. Also don't seed too low or too high number of cells, it affects their growth and morphology. I hope it will help.
good primer company in Germany
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Hey, we've been getting primers so far from biomers.net for short primers (~25bp) they are o.k. but we need a lot of longer ones ~100bp and they don't seem to be very good from this company. Does anyone of you have experience with long! primers and other companies in Germany? thanks, Anne
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Hello all ;) Our lab has ordered primers from at least 2 companies: MWG (near Munich) and Biotez (Berlin). We use primer with a length of 20 to 70 nt and had no problems with both companies. Mathias
-RT works better than +RT
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Hi everyone, When I do q-PCR I met a weird problems. In my minus RT control, there are products appeared and more surprisingly, it works even better than +RT. And in both case, the Ct value is around 27-28, which may exclude the possibility of reverse transcriptase activity of Taq DNA polymorase. I was so confused and could anybody help me to figure out possible explanation and solution? Thanks in advance. --- Eric
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Thanks for reply. Actually, I performed DNase treatment, but I am not 100% sure that I get rid of all of them. I will have a look at that thread.
BrdU assay for cos-7 cells plated onto poly-D-lysine coated coverslips
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Hi all, Hope someone can shed some light on this. I've had a nightmare trying to keep Cos-7 cells attached to poly-D-lysine coverslips during the DNAse treatment prior to antibody tagging of incorporated BrdU. The protocol I have uses a 'Mix' that is made up of DNAse1, Mg, Tris/HCl and PBS made up to 25ml with water.. but.. it also includes 2uL of B-mercaptoethanol, which I understand is a potent breaker of disulphide bonds and thus denatures proteins. The cells are previously fixed with 4%PF and permeabilised with 0.15% Triton/PBS. At the stage of incubation in the DNAse mix, almost all (>90%) of the cells detach from the coverslips and are lost. I haven't seen any other protocols where the DNAse treatment includes B-ME even at this low conc. Cos-7 cells normally, adhere very well to poly-D-lysine. As I understand it, the force maintaining the cell adherence to the slips is pretty much all electrostatic, rather than the protein-protein adhesion one would get with laminin, and the DNase mix I use is the obvious culprit in the loss of the cells (all cells are there immediately before the treatment) could the B-ME be causing this? I plan to run the assays again using 3 different protocols, including DNAse Mix without the B-ME (and a good old fashioned HCl blast) but I'm really curious -a- why would there be B-ME in the DNAse mix anyway?.. and..-b- could the B-ME disrupt the relatively weak electromag forces holding the cells on the poly-D-lysine? Thanks in advance
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Hi! Maybe a little help. I use Roche BrdU incorporation assay. I don't need fix the cells, just grow them, make the experiment, and use this kit. It's colorimetric, fast and I always got good results with this. It doesn't matter that u have attached or soluble cells. best z
Separation of high MW proteins on SDS-PAGE
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Hi There I'm trying to get good separation of high MW proteins ranging from 150kDa to 500kDa. I've been using 8% Tris-HCl SDS-PAGE with a 3% stacking gel, which I have to run for fairly long periods to allow the larger proteins to run into the gel. We use the Bio-rad proteanII minigel system, which has worked well for us so far. I see that Tris-Acetate gels are recommended for separation for high MW proteins. I'd like to give this a go, but can't find a good protocol anywhere. I know Invitrogen and Bio-rad sell pre-cast gels, but that's not an option, we have to pour our gels ourselves. If anyone has anything links or protocols or thoughts, they'd be much appreciated.
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I would try gradient gels.
THP-1 cells and 2-mercaptoethanol
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Hello! I read that 2-mercaptoethanol should be added to the medium to culture THP-1 cells. However, I will study the NF-kB binding activity and I read in some article that 2-ME should be eliminated before the experience because it is a reducing agent so it can cause some interference. The authors of the article refer that they eliminated 2-ME 72 hours before starting the experience. Does anyone have a suggestion on how to eliminate 2-ME in a more simple way?
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I do cytokine expression in thp-1 cells and I put 1.75 uL into 500mL of RPMI-1640 media. Works great!
oil red o staining protocol?
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hi, everyone I wonder whether somebody did oil red o staining for MCF-7 cell line before, now, I am doing this expriment, unfortunately, I didn't get good results. So, I need help! Thank you!!
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Any body is doing on cell line study on MCF-7, I m doing with RPMI1640, without insulin & Na pyruvate addition, not successful. Can any body able to send the protocol of MCF7 cell line study on this email sinchan35@gmail.com
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HOW TO USE THE “METHODS” GROUP:
Firstly, thanks to everyone for joining this group. The common interests in discussions about methods is reflected with the highest member numbers in “researchgate” at the moment.
However, with reaching this critical number of members few problems arise.
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So far we had the problem that redundant topics have been started, e.g. about ELISA , PCR, etc.
Threads have been moved together with common topics to avoid redundancy and this will also allow members to find topics easier due to less numbers. Sorry in advance if you can't find your last thread posted. Please search in the appriopiate topic (at the moment less than 40)
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GROUP MESSAGES:
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These will be from now on deleted. Sorry for this.
Thanks for your time. Enjoy this group and I hope you get help with your questions or have productive discussions.
All the best,
Axel
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Thanks, Axel. The new layout is much easier to navigate!
Culturing olfactory neurons: Protocols?
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Hello there, Does anyone have a good protocol for culturing olfactory neurons? For the moment, I'm trying to get my culture started in Neurobasal, but it's not that easy :)
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Dear Evelien, I do not know how many Neuro -researcher are signed up for this group. I don't have any experience regarding neuron culture. But maybe try to post a thread in "neuroscience". 330 members signed in there and I guess there are more specialised. Good luck, Axel
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7 answers
Hi everyone,
I'd like to perform whole RNA amplification of minute samples. Anybody experienced in this technique? What about biased amplification (underrepresentation of low expressed mRNAs)?
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Hi,
Thx for comment. Actually I'm thinking of disseminated human tumor cells in peripheral blood circulation. I'm completly new in this field as my expertise is on single cell DNA amplification.
Besides from being a beginner further difficulties may be the fact that I need to work on labeled cells and that I can't really get rid of the genomic DNA (due to protocol/technical reasons).
Back to your questions:
Eukaryotic mRNA
Hopeless?
Problems with DNAse treatment
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Hello, everybody! I'm with a big problem, so I registered in here to see if somebody could help me. First, sorry for my bad English... Well, I'm trying months to do a DNAse I treatment with my trizol extracted RNAs. After the treatment (with addition of EDTA, like the protocol), I reextracted the RNAs and I run an agarose gel. The RNAs are good, but when I do a PCR with the cDNAs synthesized using them, there's no bands in the tester lanes, and appears a band in the negative control lane... I always use a mix to pippete my reactions... So I'm going crazy with this problem! Please, help me to understand this!!!
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Dear Elisa. I am using Quiagen easykit for RNA preparation and further use Sigma DNase treatment on the spin columns and it works fine. I guess you checked the RNA concentration and added enough to the cDNA synthesis. Depending on your product length you might use random hexamers instead of oligo DT. That you don't get a PCR product (I guess it is a housekeeping gene you are testing) just tells you that either there is not enough RNA or cDNA synthesis did not work. Why the neg. control is pos. I can't explain. Finally, to avoid genomic DNA contamination the best thing is to design your primers spanning exon-intron boundaries. Thus this only picks up cDNA but not genomic DNA and you would not need to use DNase at all. Hope this helps. Best wishes, Axel
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Hi,
I have been using the SuperScript kit to reverse transcribe some polyA tailed mRNA... the strange thing is after purification with RNA hydrolization and precipitation, the amount of cDNA measured with Nanodrop is 7 times the amount of input RNA... I would think that something (unpurified dNTP's, pieces of RNA whatever) is absorbing at the same range... but 7 times??
Does anyone have an explanation for this?
Thanks!
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nanodrop will measure ALL nucleic acid in your sample including dNTPs, primers, RNA, DNA, etc.
You need a DNA specific assay. Pico green is good if you have double stranded cDNA. Invitrogen sell a range of Quant-it kits, the double stranded and RNA ones are quite specific but the single stranded DNA one doesn't seem to be.
Blocking cell attachment (anti beta 1 integrin)
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Hello, I hope everyone had a good Christmas and New year. I am currently carrying out some integrin blocking experiments (inhibiting cell attachment to ECM proteins) with a number of cell types and I was wondering if anybody could advise me on a suitable commercial beta 1 integrin antibody. I have tried using Millipore/Chemicon MAB1987 (clone P4C10), however this is unpurified tissue culture supernatant and I have observed an increased attachment of fibroblasts on fibronectin using this antibody! I am currently looking at two other Chemicon antibodies MAB2253Z (Clone 6S6) and MAB1959 (clone P5D2) and the AIIB2 from Developmental Studies Hybridoma Bank. Has anyone had any experience using these antibodies or could suggest an alternative? It would be much appreciated. Thanks
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Hey Sally, wish you first of all a happy new year. I am working with human adenoviruses, which are using for the cell entrance as a tertiary receptor an integrin depending process. Therefore, I know a pretty good paper, where they used P1F6 in combination with P1D6 to demonstrated that human adenoviruses are using alpha5beta 1 integrin for internalization. The paper is the following one: "Integrin alpha5beta1-Mediated Adenovirus Infection Is Enhanced by the Integrin-Activating Antibody TS2/16" ELIZABETH DAVISON,1 ROSA MARIA DIAZ,2 IAN R. HART,2 GEORGE SANTIS,1 AND JOHN F. MARSHALL2 PMID: 9223518 "inhibition of adenovirus infection was increased to 71.8% when P1F6 was combined with P1D6 (anti-alpha5), suggesting that alpha5beta1 may be involved in adenovirus infection." I hope this helps you. Best Ijad
I need proctol for sigma DNase I: I need proctol for sigma DNase I
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hi everyone, I just bought DNase I (sigma) powder from the reseller without knowing the protocol.... I wonder is there anyone who have also bought the same product as I did. Tell me how can I treat the DNase I powder, what kind of solution should I use to dissolve the powder... and how can I stop the reaction? I have tried to find the protocol for the DNase I in Sigma however without luck...
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Hey, check this one: http://www.sigmaaldrich.com/sigma/bulletin/ampd1bul.pdf you can stop the solution with phenol-chloroform extraction and alcohol precipitation, or with some EDTA.
Tritrophic interaction
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Can the interaction between plant-Arbuscular mycorrhiza (Mutualist)-pathogen be called as tritrohpic? or the term is restricted for plant-insect-predator interactions?
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Maybe try to ask this questions as well in the Group "Botany disscussion board" Cheers, Axel
strange phenomenon in msp
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I have done a successed MSP experiment two days ago, but now curiously, when I do the same experiment now, I can not amplify any band with totally same reagent, even on the positive control. Please help me to solve that. I am frustrated with that problem for long time. The following is detail: I work on the mouse genomic DNA. My prime of MSP is salt-free purification, but dissolve in the water for 100um stock solution, and diluted to 10um storage solution. my product is about 100 bp to 200bp, is very short. My bisulfite treatment is similar with http://www.protocol-online.org/forums/inde...?showtopic=9375, but I use Qiagen PCR purification kit and I do wash after the last step. especially, I use tRNA and sodium acetate but not ammonium acetate suggested by methylnick. So I am not sure the recovery rate of Bisulfite DNA is high. I have changed the fresh everything, incuding water and primer (but same stock solution), but it does not work. Additionally, My MSP is just one PCR for 40 cycles, but not nested. I think about it, and there is several possibilty: one, the primer is degraded in short time, but when I got fresh work solution, it still not work. I am wondering whether the stock solution should be degraded. Are you storing the stock solution in TE (ph8.0)? two, my bisulfite treated product is less producted, so the it is degraded very soon. I also dissovled in the water, wihich is same with protocol suggested. I don't know whether the DNA precipitation is so less effective. Please give me diganosis, thank you very much.
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Hi, I´m new here. This is my first post. I´m trying to analyze the methylation status of a promoter too. I extract DNA from several rat tissues using the phenol-chlorophorm method. Then I use the DNA Imprint Modification Kit from Sigma with 1 microgram of DNA. After that I do a MSP using primers designed by Methyl Primer Express Software. In my first PCR didn´t get any specific band (I´m looking for a 244pb band) with any of the primers of the set (primers for methylated sequences and primers for unmethylated sequences) . Then I change the reaction conditions (changing the Mg concetration, annealing temperature and decreasing primers concentration to 0.2 micromolar. Doing that I get a band in some samples and I decide to sequence that products. But the sequence I get isn´t the one I looking for. Then I thought that the primers were not specific for my promoter, that´s why I get a plasmid with my sequence to have I positive control. After that I treated the plasmid with the same Imprint Kit from Sigma with the same protocol that I used for the genomic DNA. I run a MSP with my plasmid, and I get a product with both set of primers. But when I try with the genomic DNA samples I don´t get a band of the size I´m looking for. Any suggestion...???? Thanks....
gel analysis software
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need a site for free download of a software called data analysis of bio-rad model 620 denistometer or any related softwares for gel image analysis can anyone help me thanks
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I suggest you the imageJ software. It's a free image analysis (including gel densitomeric analysis) software from NIH. easy to use and fulfill of tips...try it. bye
RBC Isolation / Purification
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I am looking for basic information regarding blood fractionation, specifically how to best isolate and/or purify red blood cells. Fractionation is easy enough by centrifugation, but I want to know how to obtain the largest yield of isolated RBCs, washed free of plasma proteins. Thanks in advance!
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Simply, wash blood sample several times.
Protein Overexpression: Cell Growth before Induction
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Hello Everyone, I have been following a protocol for overexpressing my protein but I have been getting very low yield. So instead of growing the cells until they reach an OD of 0.5-0.6 as indicated on the protocol, I have been growing them until they have an OD of ~1.7. The yield has significantly increased. Should I be worried about letting the cells grow that long before induction with IPTG (~4:30hrs)? Basically, I would like to know how long is too long when growing cells for protein overexpression. Thank you very much for your time and help.
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Well actually, according to Studier (2005) you can grow them to an OD that high with no problems. He wrote a good paper on auto expressing proteins in very high density cultures. I have been using a modified version for expressing membrane proteins and it seems to work well for me. It all depends on your media, and growth conditions.
Plasmid DNA storage
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I was wondering if anyone have any information regarding the kind of material(s) best for storing plasmid DNAs at room temperature besides the eppendorf tubing material. I tried storing the DNAs in glass vials in presence of both water and glycerol and found the DNAs starting to degrade within 3 weeks at room temperature. Any info or leads on this would be greatly appreciated. Thanks so much!
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You can put a drop of plasmid DNA on a Whatman paper and let it dry. Then wrap it in plastic film and store on RT. Any time you want fresh plasmid DNA you can pinch a piece of paper from the area where your drop did dry, wash in 50-100 ul of water and use this water for transformation. I was able to transport entire minilibrary of ~300 clones on couple of "spreadsheets" and then restored it after ~ 4 months.
advice on different Y2H kits/systems/screening services
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I recently switched from a yeast molecular bio lab to a mammalian cell bio laboratory. The new lab is currently looking to do a yeast two-hybrid (Y2H) screen. Our protein of interest is a ~50 kd integral membrane protein but we are mostly interested in its ~5kd cytoplasmic domain (as this is the domain we think is mediating the interactions). Although I do have experience in yeast biology, I have never actually performed a two-hybrid screen myself. I was wondering if anyone here has had experience in doing a Y2H screen and if they could share their personal opinion on and advise me on which commercial kit (or what lab's vectors/strains) to use. Or if they think that I would be better off contacting a company to perform a custom screen and if they can advise me on which company? Currently, I think Clontech's Matchmaker 3 and Invitrogen's ProQuest with Gateway technology are the two commercial kits that best meet the lab's needs regarding the protein we wish to screen. I appreciate any input any one can give me on the Y2H screen, including any pitfalls I may encounter. Thanks! Jennifer
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Update: I have actually run the screen and found nothing. I decided on Clontech's Matchmaker Gold system because of its use of Aureobasidin A (AbA) resistance as one of its screening markers, which is supposed to be a more stringent selection & reduces background. Instead, I get absolutely no background. I have tested on lower concentrations of antibiotic and still nothing. I have also tested on -His media with X-alpha-gal and still nothing. I have made sure I have screened an adequate number of diploids, the viability of the bait, prey, and diploid strains are all good, the baits are not toxic and are expressed. I have contacted Clontech's technical support but they seem pretty stumped too. I'm not willing to give up and say I can't detect protein interactions with my protein of interest using this method. Is there something else I may not have thought of to test?
PC12 cells do not attach on collagen
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Hi, my PC12 cells (transfected with VGlut2) do not attach to the cell surface when plated on collagen coated cover slips. Any suggestions how i can change that? I tried and tried. The grow happily in suspension ... Thanks, Tine
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Hi Tine.....Were you able to get them attached to the collagen dish? If so, do you know how to take them off of it? I am trying to harvest smooth muscle cells from fibrillar collagen with no success and thought you may know.
low unspecific products
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hi ppl...im doing PCR on human genomic DNA... and im getting lower products than expected. im trying to amplify a 3kb genomic DNA. However, my bands are appearing at less than 1kb area. i got bands mostly on d 1st 4 annealing temps. these are my reagents n conditions: PCR buffer 2.5mM(in hse): 2.5 ul Primer F(4uM): 2.5ul Primer R(4uM): 2.5ul 25mM dNTPs: 0.36ul Vivantis Taq Polymerase(5U/ul): 0.5ul 100ng/ul DNA: 2ul 25ul reaction 94 2" 94 25' *51-58 30' 68 4" 68 10" 30 cycles tq in advance
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I would suggest going for the Advantage2 long PCR kit because it is designed for longer fragments and has a better proof reading enzyme. hope this helps.
Gradient mixing
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How does gradients like pH gradients used for isoelectric focusing or sucrose gradients used for centrifugation (rate-zonal or equilibrium) not mix? Say for example the pH gradient used for isoelectric focusing. I know they are made from carrier lymphocytes and such but doesn’t mixing occur at the boundary between the two pH zones. Since pH is determined by the exchange of protons between species, why doesn’t the lower higher pH region donate some of the protons to the lower pH and therefore mess up the sharp boundary between the pH gradients? In the density gradients used for centrifugation, why doesn’t sucrose from the more dense (more concentrated sucrose region) move into the less concentrated sucrose region? I don’t understand the theory behind it, if someone could explain that would be great. Thanks.
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Yeah Wagner you're right. Some commercial sticks of pH gradient are available on the market.
Protein transfer to nitrocellulose membrane: transfer problem
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4 answers
I transferred proteins bands from an SDS-PAGE gel to nitrocellulose membrane, WITHOUT staining it first, but my membrane came out blue. When I processed the film after immuno blotting, the background was really high. Does anyone have any idea what is going on, and how can I stop this from happening again?
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The membrane was just light blue all over; it was a wet transfer @ 150V. I don't think that there was any interruption during the transfer, though it is possible that I had the thing hooked up backwards, like Tim Hucho suggested. It did transfer over though, and I got bands after tagging with an antibody, but there was lot of background as well. *shrug* Well, we'll try again and see what I get. Thank you both for your help.
problems about isolating genomic DNA by alkaline
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I am trying to isolating genomic DNA by alkaline. But I just abstract protein... I worked with tail snips from adult mice 6weeks old,are these mice too old for NaOH alkaline hydrolysis? and I don't know the principle well.We know that DNA will be dameged at high temperature,but,I found a DNA abstract method named HotSHOT(hydrolysis at 90 degree centigrade for more than 30min),genomic DNA abstracted in this method can be used for PCR.Isn't it denatured? Your reply is highly appreciated.Thanks!
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There are lots of genomic DNA isolation kits available...Promega and Qiagen each have one. They're all very easy and reliable. Tailsnips can be kept in the freezer for a very long time. The age of the mouse has nothing to do with the integrity of the DNA present in its tail.
How can I measure how much bacteria growth occurs in reused water bottles?
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I am currently doing a science project in which I have to measure the amount of bacteria growth in reused water bottles. I am not quite sure as to how to conduct the experiment. Advice on how the problem should be stated, the materials needed, and step by step instructions on how to perform the experiment are greatly appreciated. Please and thanks!
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Hhmmm.... I would put some water, shake the bottle, plate it onto agar plates and count the colony forming units after incubation at different temperatures, I would start with 28-30 degrees, this sounds like the best idea... y
cDNA dilution for efficency calculation
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Hi I'm using ddCt method for relative quantification and I want that this value is correct. I know that ddCt calculation is valid if the amplification efficiencies of the target and reference are approximately equal; a sensitive method for assessing if two amplicons have the same efficency is to look at how dCt varies with template dilution. cDNA must be diluted over 100-fold but I don't know if concentration is important. Can I start with 100 ng and then go on with 50 ng, 10 ng, 5ng and 1 ng? Or I have to start with a lower concentration to have good results? Thanks
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Hello, To calculate the efficiency of my PCR I usually make a regular PCR that I dilute over 12 logs (I mean 12, ten fold dilutions: 100, 10, 1, 0.1, 0.01, 0.001 ...) then I run dilutions from 10.-4 to 10.-12 and I calculate the efficiency of the PCR reaction using at least 5 points. In any case, 100ng of template will be too much DNA (it will inhibit the PCR reaction thus giving you a bad estimation of the efficiency). I would say starting at 10 ng will be fine. I hope it will be helpful. Regards, Emiliano.
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3 answers
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Both USDA Labs and FDA have this data for Co-enzyme A and Co-enzyme E in milk and dairy products.