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Questions related to Molecular Biological Techniques
My streptavidin ELISA is producing very high OD values for both my positive and negative sera. I have purchased commercially available streptavidin coated ELISA plates that have been pre-blocked. The protocol I am using is:
Initially washing the plates - incubating with biotinylated peptides (2 hrs) - blocking with PBS + 2% BSA (1 hr) - addition of primary Ab (bovine) diluted in PBS (1 hr) - addition of secondary Ab (rabbit anti bovine) diluted in PBS (1 hr) - each of these steps were performed at 37 degrees C and washing was performed between each step using 0.0125% Tween 20 in Saline.
Are there any suggestions on how can I reduce the high background observed across the plate and especially in my negative control sera?
Thanks
Does anyone have a tried and tested protocol for intracellular staining of RBC's. I have tried a few methods, but the RBC's all seem to disappear during the staining step!
I ligated a DNA size of 3.8kb into topo TA vector. I expected a band size of about 7.7kb from my agarose gel electrophoresis after plasmid purification but got a band size of about 5kb. Pls what does this band size signify or is topo vector less than 3kb?
I'm using the CRISPR/Cas9 gene editing method to target a gene in human iPSCs for mutation correction via the homology directed repair pathway. I want to compare the efficiency of different gRNAs to determine which gRNA will be the best to use for this application. Normally, this can easily be done using a genomic cleavage detection kit or a mismatch detecting endonuclease, followed by visualization of the resulting fragments on a gel; however, the mutation we are trying to correct is heterozygous in this particular patient. Since mismatch endonucleases, such as the surveyor nuclease, are sensitive enough to detect a single bp change, would the heterozygous nature of this SNP lead to a overestimate of the level of cleavage occurring?
Has anyone else come across this issue? If so, does anyone have suggestions for other methods to detect cleavage events?
I infected the cell lines H1437, H2073 and H2228 with a lentivirus that express resistance to puromycin. Does anyone knows the best dosis and time for selection? I have found information using 2 micrograms per ml for 3 days, but when I did the kill curve for H2228 it did not seem to be enough.
Any information or experiences would be greatly appreciated!
Thank you!
We are trying to clone into a plasmid(pBUDce4.1) with Zeocin resistance, but are finding it very difficult. The Zeocin appears to have very mild selectivity (i.e. transformed cells grow only slightly faster than untransformed cells on Zeocin agar plates). We have now managed to select transformed colonies, but when we grow up liquid cultures there is very little plasmid post extraction on agaros gel .Is this poor selectivity something seen by other people (I am aware Zeocin is unstable in high salt, light etc. so I am using low salt (5mg/l) LB and trying to keep out of direct light)? Any other thoughts as to why this isn't working for us?
My sample is filtered and concentrated liquid and I'm using Qiaamp viral RNA mini kit to elute viral RNA.
Since this kit is not designed to separate viral RNA from cellular DNA I used RNase-free DNase set (Qiagen) for DNase treatment. I pour 80ul of DNase to the column for 15min. at room temperature before the washstep (AW1 and AW2).
260/280 of the eluted RNA was too high.. it was almost 3.4.. and it is also very high even when I didn't do DNase treatment.. I have no idea why this kind of result shows up.
I will be running my first luciferase assay and the protocol I am using does not specify what filters or filter settings I should use. The plate will be read on a lumometer from Biotek, Synergy HT. What would a general filter setting be to measure the luminescence?
Does anyone know how to make the fluid to remove the coverslip of glass bottom dishes?
I am performing a KO of my gene by frameshift in zebrafish. I have been screening this by the disruption of a restriction enzyme site in a PCR fragment. I now have an incross of 2 mutant fish and am hoping raise the progeny and get some homozygous fish. I will use DNA from tissue sample as the PCR template but I am wondering how I can separate homozygous from heterozygous. In my experience so far the F1 generation from an outcross with wild type showed an extremely faint undigested band (wt DNA) compared to the really bright digested band (indicating mut DNA) for some reason even though an outcross should automatically result in heterozygous fish containing both wt & mut (digested & undigested) DNA. I presumed that the concentration of the mut & wt allele would be similar. Has anyone come across this difficulty before. Could there be a reason a mutant would amplify more in a PCR reaction (it is only 7 bp shorter)? Any suggestions for a way to tell the difference? I am concerned since the band is so faint on heterozygotes I may misidentify heterozygotes as homozygotes.
Hi All, I am transfecting two vectors (one gRNA vector and a HDR vector) to KO a gene. I have puromycin resistant in HDR vector. I am using Fugene for transfection purpose. I was wondering to know how long should I wait to start selection after transfection of the crispr vectors?
I am planning to work with Addgene's pLKO-tet-on plasmid. I checked the protocol on the website and comments left here so far. I am a bit confused. Should I design the oligos exactly in the protocol (5'-CCGG for AgeI and 5'-AATT for EcoRI)? Because it is one bp missing in both RE sites and it results in oligos which have mutant RE sites. If they are mutant, how will the ligation work? I would be really happy if someone could explain me. One more thing, I came across so many people who had troubles of getting positive clones after ligation. I am wondering what is the latest situation. Is there any tricks that I should know? Thank you very much for your answers in advance.
I have generated the lentivirus particles with my GOI. But did not get transduction efficiency in Jurkat cells post 72 hrs why? any suggestion ?
Particulas: used 24 well plate :
50,000 cells with Polybrene 8ug/ml
Thank you in anticipation
I am working on unknown large plasmids (50-500 kb) that I need to characterize. I plan to use the Large Construct kit for extracting them and remove chromosomal DNA.
1) Will I see large plasmids on a gel???
2) Is Primer Walking a good approach?
Is there any other methods less time consuming?
NGS will be done but I want to confirm using Sanger Sequencing.
Thanks,
I've been trying to perform a Northern blot for a while now, with no positive results.
I use Digoxigenin labeled PCR products as a probe (after boiling for 5min) in hybridization buffer (50%formamide + 5xSSC, 50mM Na-P, 1%SDS, 10ugssDNA/ml, 5xDenharts) @42*C o/n, dollowed by wash, block in 2%milk 0.3% PBS-T, and incubation with an anti-Digoxin antibody.
I know the transfer works, and that RNA is present after prehybridization (via methylene blue), I also know the antibody works due to dot blotting the DIG-labeled probes.
Does methylene blue staining inhibit probe binding?
Every time Ive tried N.blot Ive stained my membrane with methyl blue before in order to see whether the transfer worked. It's all I can think of at this point, otherwise it has to be my probes.
-CP
I am analyzing samples transfected by CRISPR using the T7 assay. And when I am trying to analyze it on the gel, I can found smears on my samples. I don't know if I added too much enzyme or very long incubation time. Any suggestions?
Here is my recipe/protocol used.
purified PCR product -150ng
NEB buffer 2- 1uL
Water- 9.5uL
Reaction 95C-10min, 85C- 5min (0.1C/sec), 65C-2min (0.1C/sec), 45C-2min (0.1C/sec), 25C-hold
Add 0.5uL T7 enzyme. Incubate for 1hr. Stop reaction by adding 1.5uL 0.25M EDTA. Run on gel.
thank you.
Reaction:
Is there any affinity of microRNA to glass surfaces? I tried to homogenize brain samples in order to get maximum yield of microRNA.
Hi I am looking for any alternative protocols to purify TMV virions, other than the commonly used PEG precipitation.
Two main problems that I have with PEG protocol is that,
1\ any macromolecule will co-precipitate as well
2\ so that PEG cannot purify full length (300nm) TMV virions from other shorter rods (probably breakage), and other small discs. (thanks to people responded to my earlier question)
So I am wondering if there is any purification protocol or method that can get mostly the full length rods and rid breakage ones and discs?
Thanks a lot in advance
I am trying to isolate a nuclear protein NF-kB from raw cells in order to do a western blot.
I have searched some protocol for nuclear isolation and finally came up with one. But in that protocol, apart from adding 420mM Nacl for NE buffer , they have added an extra 400mM using 5M NaCl directly onto nuclear pellet and again 1 pellet volume of NE buffer. Can anyone tell why extra NaCl should be added, and if we add double the volume of NE buffer, won't the protein get diluted? I'm attaching that protocol along with this.
i am new to enzymology and want to study the discipline through history based books (i,e, how was the Kreb's cycle discovered, methods used then, etc). what are your suggestions to achieve this?
better to be from wiley or springer.
Hello, I have developed a Surface Plasmon resonance sensor using LED of wavelength 635nm and CMOS webcam as source. I am using the diverging rays of the LED as the change in incident angle. When I put silver coated glass slide on the prism I get a dip at a particular angle. I have test the sensor by immobilizing with MUA , EDC/NHS and IgG. The sensor can detect the shift in angle for all the layers. But when I put liquid dielectric medium like DI water, BSA or PBS buffer the shift disappears. I can monitor real-time data with the webcam and so when the liquid sample is passed I should be able to detect the shift. I have attached the file of how the dip looks like.
I am having a rather odd issue with a ligation procedure. After a (hopefully) successful ligation of a 1kb insert into a 6kb vector, I transformed some Top 10 cells and got (very few) colonies on LB+Amp plates, but always more than the no-ligation control plates (indicating a hopefully successful ligation). I screened several (5-10) of these colonies and isolated DNA using a mini prep, after which I linearized all the DNA and ran on a gel. At first, I got really fuzzy bands, both in the samples and in the ladder, so it was hard to distinguish if they had any insert in them. Also, the unligated vector ran at a different size than expected, and some of the samples had two bands in them or ran faster than the unlighted vector. Overall, it seemed like a very messy gel, so I re-optimized my gel conditions to make sure I could at make any conclusions from the DNA sizes (made new buffer, made sure the loading was done correctly, lowered the voltage to use 90V for 2 hours using a 0.7% gel). After this, my ladder looked crisp and properly separated, and my unligated vector (linearized) ran at the right length and also looked pretty clear. However, all my ligated samples had absolutely no product in them at all! For the first gel, I used 500 ng per well, and all the wells looked equally bright and with similar amounts of DNA. Noticing this was a lot of DNA to run, I reduced it to 125ng of DNA, which showed up perfectly fine for the vector but not for the samples. There is no smear so I don't think it's degradation or nuclease contamination, so I am not sure what to do next. Any ideas? Thanks! I am attaching an image of the gel, with the only visible band being the linearized unligated vector (6kb), and all other wells being my ligation products. Any help would be greatly appreciated!
Hi,
For the FreeStyle HEK 293F cell serum-free suspension culture, is it possible to use another media (a cheaper one) than the FreeStyle 293 Expression Medium?
Thanks.
I expect a 70 bp amplicon which shows up only with 2% (or higher) DMSO in the PCR. But with DMSO, the size of this amplicon goes from 70 bp to ~110b bp on 3% agarose gel. Same happens when a positive control PCR is done using same primers. In the positive control too, the size goes from 70 bp to ~110 bp when done with 2%DMSO. Is this size shift common if DMSO is added in to PCR?
Hi
I would like to ask about protein precipitation by salting out method.
I'm going to precipitate collagen from fish scales. Some articles said the supernatant of the extract salted out by adding NaCl to a certain concentration, for example 2.0 M. Does it mean adding solid NaCl or NaCl solution? And how to calculate it?
Can you explain it more detail?
I've read ebook from Coligan (2001), Current Protocols in Protein Science. The book explain about salting out method and the calculation. Can I use that equation to calculate the mass of NaCl?
(The equation on the attached file)
Thanks!
I have a stack of 96 well plates containing lysates in RLT Plus. I dont want to process them all at once, rather a few wells per plate to start. Will re-freezing the rest of the plate damage the samples?
Does anybody have a lot of experience with Image Studio Lite by LI-COR and quantitative Western blotting?
I am using Griess lysis buffer for cell lysis which contains DTPA & EDTA. Will that interfere with CuCl2 when detecting RSNOs in the cells? How to overcome this?
Hallo everyone,
I've been having some trouble isolating bacterial RNA from a gram positive organism for a RNA Seq analysis. My problem is that I always get a very intense "cloud band" on the agarose gel around the position where the 5S RNA band should be.. I've tried several protocols and kits, with and without bead beating, Trizol, Lysozyme, but it happens every time.. The first idea was that these are products of degradation, but then again the intensity of the 23S and the 16S bands clearly remains very high. And also, on a Bioanalyzer this 5S band definitely does not look like degradation, but rather as a sharp peak around 127 nt.. Does anyone have any experience with that? If this is in fact the 5S rRNA, why do I get such accumulation, how should I get rid of it and would it temper with my RNA Seq results?
Thank you all in advance!
The purpose of denaturation is the break down of dsDNA, so what is the need of intial denaturation while denaturation can do the same.
We would like to dephosphorylate purified proteins (in vitro). How do we decide what type of alkaline phosphatase to purchase?
We are recently trying to isolate RNA of FACS sorted cells with a population of 100K. The cell population we are sorting is very sensitive and we noticed a lot of cell death after the sorting procedure. For RNAisolation we are using RNeasy Mini Kit from Quiagen. Although we tryied to collect the cells with different conditions (PBS, RLT, BSA coated falcon), we did not manage to isolate any RNA. Our unsorted population however gives us a high RNA yield.
Has anybody had similar problems and has a solution we could try?
I'm in the initial stages of planning a miRNA seq experiment using human cultured cells and decided on TRIzol extraction, Truseq small RNA prep kit, using an illumina HiSeq2500. The illumina webinar suggests 10-20 Million reads for discovery, the QandA support page suggests 2-5M, and I wrote the tech support to ask, who suggested I do up to 100M reads for rare transcripts. Exiqon guide to miRNA discovery manual says there is not really any benefit on going over 5M reads. I was hoping to save money by pooling more samples in a lane, so I was hoping someone with experience might be able to suggest a suitable number of reads.
i got mann whitney test value U=1402, i am confused about this so large value. this value is right? i mean if value comes in thousands then no need to worry or there is any problem??? please
I'm going to view localization of certain fluorescent protein in Enterococuccus. As they tend to form chains, and for analysis I need to use single cells images, I wonder if I can fix in first with PFA and then proceed with microscopy? Are they still considered alive?
Another question is if I want to track the appearance of my protein on the cell surface during the cell growth, may I use agarose pad, or it wouldn't divide on it? Can I use BHI agar instead or it will interfere with imaging due to less-transperent properties?
That's where fixing becomes a problem, once fixed will I be able to track division or not?
Hello everyone!
I am using AB Stepone system with Qiagen QuantiNova PCR kit. After doing my first trial, my qPCR results were very strange, the plot are "hook"-shaped (attachment). Anyone know what happen? The machine I use is not calibrated (the last calibration was around 2012~2013), will that cause any problem like this?
Our plate centrifuge are out of order and I cannot centrifuge properly, but no visible droplets remains on the wall. There are still some bubbles on the top liquid surface, is it a possible cause for my results (I thought the bubbles will be removed upon heating)?
I am doing a quantification experiment with 5 standards (300,000 copies, 1:10 diluted to 30 copies). I am new to qPCR and really confused by the result, please help :(
Thank you for your help!!
Hello everyone!
My protein which has a GFP tag is well expressed in nuclei. To visualize cell boundaries in different layers of the root sample, I am planning to counter stain my samples with propidium iodide and DAPI.
I will be taking images using 3i spinning disc confocal microscope.
I want to know if anyone has a working protocol for such settings.
Earlier, I tried Hoechst 33342 to stain nuclei but it appears that even incubating for 30 minutes, the stain was not able go inside the cells as the cell boundaries were clearly visible during microscopy. I tried DAPI as well, but the background was very high even after 3-4 washes and the intensity of nuclei was not great.
I will appreciate if you could share with me your experiences and suggestions regarding my question.
Thanks!
Does anybody have experience with ACC (Acetyl Coenzyme A Carboxylase) and pACC detection by western blotting? It is often used to confirm the status of AMPK activity. The antibodies we use are from Cell Signaling Tech. and they are cited in several publications. However, our anti-ACC does not work at all, while anti-pACC recognizes an unspecific sharp band at 100 kDa and a signal, which is running around the expected MW 265 kDa, not as a defined band, but as a “diffused zone”. It should be pACC, because its intensity mirrors that of pAMPK. I am not sure, if it is a problem related to our SDS-PAGE 6% gels or blotting or lysate preparation (RIPA buffer + protease and phosphatase inhibitors, then treated 5 min at 95 °C in highly reducing conditions (beta-mercaptoethanol, 5% in lysates). We are trying to detect ACC in HepG2 cells and rat liver lysates. This is really frustrating, because according to several publications pACC and ACC should be sharp bands. Please help us to identify the critical problem and get nice pACC/ACC blots.
I want to make point mutations/corrections in iPSCs.
Which iPSC vectors would have a high efficiency?
Puromycin selection may be better than GFP/FACS sorting?
I'm at the moment trying to overexpress a protein known as amidohydrolase from Rhodococcus into e.coli BL21/DE3 using pET28a vector. To confirm the expression, I did western blot with a Anti-6X His tag antibody (given that there is no antibody specifically for amidohydrolase). Surprisingly, my negative control, which is the cells transformed with pET28a without my insert, gives me strong signal at 40KDa. Has anybody encountered a similar situation? Does anyone have an explanation for this?
I had made a cDNA library with infusion smarter cDNA library kit using Stellar Electrocompetent cells, 7 months back. Now when I grow white colonies in LB -Amp they are slow growing and don't achieve requisite culture growth. Also from them I get no plasmid when I proceed.
We are using mouse muscle DNA but ideal sonication condition is elusive. I wonder if anyone used Misonix S 4000 to fragment DNA into 500-1000 bp for CHIP?
Do I need to use DNA ladder or it is not important? what is the programme used for analysisi? and also if I have many samples could I make two electrophoresis gels or they must be in one gel?
Hi everyone,
I am trying to improve sustainability in my institute and I realized that there is an overuse of MilliQ water. I believe that for most of the operations done in a microbiology lab (buffers and bacterial media preparation, PCRs, restriction reactions, ligations, DNA assemblies, ...), using distilled water might be sufficient. I was wondering if someone has an overview of which water can be used for which protocol and maybe a list of the operations which really require MilliQ water.
Thank you very much in advance for your help!
Best,
Filippo
I am having issues with unexpected flourescent bands occurring in my negative control samples (heat inactivated / minus telomerase samples) whilst using the TRAP assay to look for telomerase activity. I have ruled out contamination, run a temperature gradient for annealing temperatures and varied cycle numbers but this had no effect. I have contacted the assay's producers but they gave no useful suggestions. Has anyone else had the same problem?!
Can anyone share a protocol for a ferrozine assay for tissue samples that is reproducible, including the lysis protocol? Also, what is the minimal amount of iron detectable using this protocol and which method was used to quantify the tissue amount (by protein?)?
I have been working on a TBI mouse model and looking at AQP4. The literature provided by abcam shows the western blot data to have a band at 46 kDa and another one at 20kDa that they cannot identify. My blots have a double band at 48 and 46 kDa and a large band at 20 kDa. Does anyone have any experience working with this antibody? Any suggestions or ideas about what these bands represent?
Hello. I am currently developing a protocol for a neutrophil phagocytosis assay using flow cytometry. We will use a GFP-expressing bacteria. Can anyone tell me if the addition of trypan blue will quench the GFP-signal of the non-internalized bacteria? I know trypan blue is used to quench the signal of fluorescently labeled extra-cellular antibodies, but I wasn't sure if it would work if the fluorescent signal was coming from protein inside the bacteria?
I am using Dot Blot study to detect 5hmC level. Dot blot detection is ok but when I use methylene blue stain to check the control, the dots are not so clear. Very faint color I can see on the membrane. Two methylene blue staining methods I used but result is same.
Method-1: Membrane is washed with miliQ water and then treated with 0.04% methylene blue in 0.5M sodium acetate (pH 5.2) for over night.
Method-2: Membrane is washed with miliQ water and then treated with 5% acetic acid solution for 15 min at room temperature. After that treatment with 0.04% methylene blue in 0.5M sodium acetate (pH 5.2) for over night.
Is there anybody who can give some valuable suggestions that could help me to solve this problem?
Our group is looking at moving away from traditional western blot methods as there are many more time efficient options now available. I have seen an impressive demonstration of using a few ml of "1-Step™ NBT/BCIP Substrate Solution" to detect protein bands on the membrane. However, I have heard that this technique is limited in that it does not allow for stripping and re-probing with different antibodies. Does anyone have any suggestions as to how this can be overcome? Or are there any other limitations that I should be aware of?
Any advice would be appreciated.
After rounds of washing and antibody staining, the protein ladder seems to fade. Does anyone know why?
Hello, I am running an Optiprep iodixanol gradient density to isolate my exosomes. With each run I also have a control which I exact 12 fractions and from the protocol it said I need to calculate the density of each fraction so I will know in which might be my exosomes.
However, although it might be quite straight forward, I am having difficulties in calculating the density of each fraction. I have been given the coefficient of extinction 320 L g-1 cm-1 and the wavelength 244nm. And with that I dilute each franction to 1:10000 and read the absorbency. However, I do not know how to get to the density from there. Any suggestion?
Thanks
I was reading SDS PAGE and methodology it was mentioned, I searched it online but i couldn't find the answer.
I ran samples (looking for c.bovis in swabs and tumors) using the same primes/probes, Taqman Master Mix, DEPC H2O that was used last week on samples that worked fine. These new samples that should had been negative tested positive for c. bovis. I replaced all reagents and reran the samples. Still positive. Some samples are negative and the NTC is negative so I do not thing contamination is the cause. I can not think of any other cause for the false positives. Can anyone offer any assistance? Thank You.
I have also included an image of the results for better visual.
Hi,
I'm trying to disrupt some genes of the genome of E. coli. For achieving this, I'm following the protocol of the P1 phage in which this phage transfers genetic material from one strain to another. The recipient strain is from the keio collection, so I don't have any problem with the selection of the colonies which were succesfully trasduced.
But after the confirmation of the disruption with the antibiotic screening and PCR, I tried to remove the Kn resistance using flippase. The flippase is a recombinase that recognize FRT (Flippase recognition target) sites and removes all the flanked area.
This plasmid must be electroporated and then grow it at 30°C, because it has a temperature sensitive ori.
I have done all of this but I have not obtained any transformant. Do you have any clue of why I don't get colonies? Can you give me some tips?
Hello
Today i want to ask about whether there is optimal condition of centrifugation
(speed, time, and temperature) to obtain cell pellet.
I have already searched many protocols, but i only can find those mentioning about bacterial culture.
What i want to ask is about the optimal condition of centrifugation to get pellet of HUVECs. For me, i collect HUVECs to 1.5ml tubes with 1X PBS and I spin down the samples with small centrifuge for about just 30 seconds.
Then i can see the pellet at the bottom.
But i learned that there are numerous conditions of pelleting down cells
so i want to ask whether my protocol described above is ok
Thank you very much!
When determining titer of T7 phage by plaque assay, I get a gradient of plaques where one side of the plate is clearing and the other half of the plate is a lawn of bacteria. Anyone experience this issue?
there are many methods to mtDNA extraction from human blood , what about the positive control used as marker in gel electrophoresis
Every time I try to isolate DNA, it ends with a smear like band on gel, or the pellet obtained is very little. Currently I am using a protocol which requires 2 days to isolate DNA. If possible please suggest a protocol that enables isolation within the same day.
Which is the best method and software for Microsatellite markers analysis?
Urgent for help!!!!!
I want to elute proteins fron Aminophenyl-m7GTP (C10-spacer)-Agarose(Jena Bioscience).
I searched some articles and some answers on Research Gate, there are many different ways. I want to elute it with sample loading buffer,and then running SDS-PAGE gel.
I want to know how can I wash unbinding protein away from the Agarose? Do I need to check the concentration again after I add same volume of loading buffer to the Agarose?
Can some tell me detailed procedure to do this?
Thanks advance.
Hello everyone,
I am trying to clone 4 inserts (816 bp, 864 bp, 1226 bp and 1699 bp) into Xho I digested pBSK (+) vector (2598 bp) using NEB's Gibson assembly kit. All the fragments used are gel purified and have a good yield. I have set up reactions by taking 0.025 pmoles of vector and 0.05 pmoles of each of the inserts, with a total reaction volume of 15ul. The reaction is incubated as per the manufacturer's instructions. I have been checking my assembly on the gel before I transform it into XL-1 Blue competent cells and I observe a faint band of expected size (7.2 kb). However, I am unable to get any recombinants post-transformation and end up with only self-ligated vector. The vector has been XhoI digested, gel extracted and treated with antartic phosphatase to prevent self-ligation. Can someone suggest tips to drive the reaction so as to achieve more efficient assembly of the fragments?
Thanks a lot!
Kajal
From the standard text, I found that ethidium bromide intercalates the double stranded DNA, then how single stranded DNA can be visualized by ethidium bromide? I am curious about different visualization methods and mechanism?
If I vortex stock primer for PCR strongly, will it break easily?
I'm trying to find the best way to measure acetylcholine (ACh) levels in brain areas of interest. I've used some commercially available kits but the results are not great (maybe problems with detection of ACh).
I need to know if you can induce gene expression by injecting it into the mouse. There are plenty of examples of using the system in cell culture but I wasn't able to find anything about using it in animals. Thank you.
Hii
I need help. I need to send some RNA samples to my service provider in US. I am planning to send my samples in Ethanol and Sodium acetate precipitate form. But my issue is I dont have an option to send in DRY ice or cold condition. Can anyone tell me will my RNA in ethanol and sodium acetate be stable at room temperature?
I know, until there is no RNase in my samples, nothing can happen. As we all tried till now is keeping in -80C until use, But still anyone tried to keep the RNA in ethanol and sodium acetate at room temperature for more than 24hr? I know i may be asking a stupid question, but accidents can happen always in someones life. Like, forgetting to keep the samples in -80C, something like that happened to anyone?
I need answers from experienced hands so that with confidence i can send my samples in room temperature in ethanol and sodium acetate.
I have been trying to arrest immortalized MEFs using double thymidine chase, I have used two different protocols, in one I added 2mM thymidine for 15 hours released 10 hours and back to thymidine for 17 hours, but when I took the cells to flow cytometry I cannot see a nice peak at the beginning of S phase instead I saw an increase of cells on S phase indeed but I still can see a G1 peak and some cells on G2/M, has anyone arrested these cells or has any suggestion? Thanks,
Is there any DAB staining method for ROS detection in pea leaves??
The storage buffer of the RiboLock RNase reagent contains 50% (v/v) glycerol that prevents the overdrying of RNA. My question is what if we would use ethanol along with glycerol for DNA drying too.
Тhank you.
I had done PCR in two batches. I had casted a agarose gel using two combs. I had loaded few samples from the first batch (most of them had been already visualized and had given good amplification) and then ladder and the few samples from second batch in the upper well. The remaining samples of second batch were loaded in lower wells of same gel.
After visualizing the gel under UV no bands were seen in the upper wells except the ladder while good amplification was seen in lower wells.
Why it occurred?
Dear all, I am currently using Qiagen miRNA profiler plates (catalogue number: MIBT-659ZF-24) to identify deregulated miRNAs in cattle under certain physiological condition.