Questions related to Molecular Biological Techniques
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!
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?
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)
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?
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.
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?
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!!
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.
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 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!
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?
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!
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.
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?
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.
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?
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?
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.
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?
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!
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?
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.
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,
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.
I have ordered the product and the product arrived as well, however I did not find any product manual and the plate layout of that particular product in the internet page of the company. If someone used it before can you please provide me the plate layout.
Thanks in advance.
Hi, question about cloning. I tried to do a massive cloning experiment, but when I ran PCR to test for positive clones, only a very small percent of the transformations were successful so back to the beginning unfortunately. I was going through troubleshooting issues and realized that "too much ligation reaction" was a reason for low efficiency. I used 5 uL of my reaction rather than the suggested 2uL that my protocol called for, thinking that I wasn't going to use it again so why not? I think this might have been my problem. Can anyone tell me why using too much ligation reaction would be a reason for poor and low cloning efficiency? Thanks!
TOP10 bacteria supposed to be ccdB sensitive. I transfected them with a gateway plasmid and the selections does not work for me.
can anyone give me some hints? Thanks.
Does anybody have experience visualizing intestine whole mounts by confocal microscopy (IF) and could give me some details about the protocol to get clear images of crypts and villi?. In my hands, the tissue is too thick and antibodies cannot penetrate so well, besides the fact that is very difficult to focus on the microscope.
In NHS/EDC protocol, there are two buffers. coupling buffer and activation buffer. (+washing buffer) And, I will conjugate PEG-COOH to amine-modified oligonucleotide.
In the NHS / EDC reaction protocol, the composition of the coupling buffer is shown but the composition of the activation buffer is not shown. In addition, all of these protocols are protocols for the conjugation of amine to -COOH in proteins, and there is no mention of what buffer to use when attaching an amine modified oligonucleotide to -COOH.
1. If the pH is kept between 8.5 and 9.5, does the activation buffer have a similar pH? What is the composition of the activation buffer?
2. In the case of the coupling buffer, the pH of the protein in the protocol is 6.0, which is higher than 7.2. Is it okay to use it as a coupling buffer for oligonucleotides (for immersing PEG particles)?
ps. If you have an EDC / NHS protocol for conjugation of the amino-modified oligonucleotide you have with -COOH (which is better for polyethylene glycol-COOH), I would appreciate it. Other reagents can not afford to buy.
I'm wondering about how RNA later works when DNA is the end goal. I am planning to culture some bacteria and send the pellet for genomic sequencing, and I was told it's ok to send the pellet in RNAlater instead of freezing it (I don't have access to liquid nitrogen and they would prefer not using glycerol).
I haven't found any clarifications about this, so I wonder will DNA really be preserved well, especially if it turns out the pellets will stand in RNAlater solution at room temperature for at least a few days.
the protein will be measure at absorbance of 280 nm and I can know the concentration of it in mg/ml using the extension coefficient but how if this protein is conjugated with HRP, because the HRP will be measure at this absorbance spectrum??
I am working with the Agilent 2100 Bioanalyzer, Small RNA Kit and with the software of Agilent 2100 Expert version B02.08.SI648. I obtained results of my last run. However, I do not how to change the scale of (s) to (nt) in the electropherogram. I tried with several options in the software, but it did not work.
We have tried various modifications to our standard Western Blotting procedures now trying to detect CHOP (GADD153) in our human cell lysates after e.g. stimulation with 1 µg/l tunicamycine, but to no avail.
Do you have some helpful pointers as to how to detect CHOP?
We have used nitrocellulose and PVDF membranes in the past. Primary antibody is the recommended Santa Cruz monoclonal mouse anti-GADD153 (B-3) at 1:250. For technical reasons, we don't do the transfer on ice, however, actin signal is always strong on our membranes.
Is it a matter of amount of protein loaded? We have done roughly 20-30 µg/lane so far.
Thanks for any good ideas or working protocols!
I am planning to use the Qiagen RT2 Profiler PCR array for signaling pathways. Is it really necessary to use the RT2 SYBR Green Mastermixes and the RT2 First Strand Kit with the profiler array? Can I use Applied Biosystems SYBR Green PCR mastermix instead (Cat no. 4309155) as well as my already synthesized cDNAs? The platform is Roche Light Cycler 480.
Looking forward for some help. Many thanks.
I have done cloning for years and just recently I have encountered this problem. When I do my PCR screening with Taq, I never run along a negative control because I know what size I am looking for. However, this time, I am looking for a band at 158bp. I am afraid that cannot tell the difference between PCR product and primer dimers, so I run a negative control along it (template that primers could not amplified). I saw a band exactly the same size as positive control.
So, I did it again without any template and I still got a band same as positive control. So I changed my water, buffer and make fresh primers. But I still have the same result.
Are those just primer dimers running the same size as my PCR product? My primers are 25 and 27 bp. If those are just primer dimers, how can I tell the difference from my PCR product? I am running it on 2% agarose gel and I used 100bp DNA ladder.
I performed agarose gel (1%) for PCR products of COX-2 from saliva samples of patients suffering from periodontitis. I could not see any bands and therefore in order to check if the RNA was isolated appropriately, I also performed agarose gel electrophoresis and again could not see even a band of RNA. Can anyone suggest where we are making mistake?
I'm trying to get subcellular fractions (specifically, I only need the nuclear fraction) from neuronal tissue. However, all my current samples were flash frozen and stored in -80C subsequently (for whole cell lysate, which was my original intent for the samples).
Does subcellular fractionation for nuclear protein require fresh tissues and cells? Will flash freezing might cause ice crystals to form and pierce the nuclear membrane, so I wouldn't get good separation?
Thank you in advance.
I designed four primer pairs and i want to set up the PCR reaction conditions (Temperatures and numbers of cycles) for these primer pairs .. is there any recommended software or programme to do this job?
The ThermoBrite Elite system automates the pre- and post-hybridization steps in FISH testing.
How efficient is it? Is it a closed system? Is it FDA approved?
Is there any better option than these cell lines to study oligodendrocytes in vitro?
I need to create hyperglycemic and hypoglycemic condition. Then I aim to inhibit perticular protein by adding inhibitor to the culture to understand effect of that protein on oligodendrocyte survival.