- Savita Shah added an answer:1Is it possible to refill the Novex Precast SDS-Page Gel-Chambers?
I never used them, but we have them in our lab and i wanted to reuse them once they're out.
No unfortunately you cannot..
Easier and cheaper to make your ownFollowing
- Lori M. Kelman added an answer:9How can I make SDS-PAGE gels that run faster?
Currently we use novex 4-12% bis-tris gels from life technologies. We can run them at 200V in MES buffer, which takes 35 minutes to run.
I was wondering if there was a protocol to hand make gels that would run faster than conventional hand made gels (i.e. less than an hour).
Does anyone know what is in the composition of these gels that makes them run fast and give good resolution?
Why do you want to run the sample faster? I think you're about at the limit with what you're doing - you might be able to run the gel faster in the cold room with a recirculating cooling system of some sort, but you will probably lose resolution.
If you want faster results, and if your lab has money, something like the Agilent Bioanalyzer (lab-on-a-chip, microfluidic analysis) will give you fast results, but it's much more expensive than a PAGE setup.Following
- Kholoud S Ramadan added an answer:5Problem with PFGE - Why is my DNA smearing?
I am attempting to work up a PFGE protocol for a Gram Positive cocci that is of interest to our lab. However, I am still getting a lot of undigested material stuck up in the loading well. I originally assumed that the SmaI digest was incomplete but we discovered it was the concentration of Lysostaphin relative to the cell suspension. Once we increased the Lysostaphin, we started to see faint bands however now all we are getting is smearing! (see attached) plus a lot of DNA still stuck in the loading well. Does rough handling of the cell suspension or delay in embedding the cells in agarose cause smearing? Or is there something wrong with the electrophoresis tank temperature or buffer temperature and the DNA is degrading? Or buffer, tubing, tank contaminants? Thanks in advance.
no problems with the smear if the concentration of DNA I'd high.Try to make digestion with restrictions enzymes and repeat PAGE if there no smear so your DNA is ok but if the smear Is still present after digestion so you have a problem in DNA idolationFollowing
- Christian Q. Scheckhuber added an answer:2Should I store the Tricine gels in an annode buffer or can I use the running buffer from the Laemmli system?
I'm wondering how other people store their handcast Tricine gels. The Laemmli gels we make are usually stored in a container of running buffer. Should I store the Tricine gels in annode buffer? Or can I use the running buffer from the Laemmli system? It is only for a short period of time (1-2 days).
I've heard some people wrap their gels for storage but we only place them unwrapped in buffer. Could that be a problem?
You could store the gel cartridge in a plastic bag and put it in a fridge. If you like you can wrap it in tissue paper that has been soaked in cathode buffer. This way, it will not dessicate.
- Jack Erron Haggard added an answer:6What's the meaning of appearance of DNA bands on gel electrophoresis of negative check wheat cultivar "Morocco" ?
Morocco is known as a wheat cultivar free genes for rust resistance and usually use as a negative check in DNA marker assay
Are the smears high-weight or low weight?
Smears not caused by contamination (in the template, master mix, or gel lane) are generally due to nonspecific amplification. This might occur if you have poor quality or degraded template DNA or if your PCR conditions were such that they allowed the primers to bind to non-target sequences.
Is your template DNA for the positive control of high quality? Have you checked it by spectrophotometer?
Is the smear for the positive control similar to what you observe for the water blank control? Is your water clean and sterile?
What is the concentration of template DNA in your reaction? What is the concentration of each primer? Overloading template can cause nonspecific amplification.
Is it possible that the positive control reaction was subjected to different conditions than the other wells? I mean, could it have been in the corner of the block, not seated properly, etc.? Have you repeated these reactions with the same results? Have you tried running the positive control in a different well?
For dominant markers, I would recommend running them in separate reactions (and gel lanes) until you are certain they work well and you know what to expect.
Based on your description of the results from the two dominant markers, I would interpret them to mean that none of the varieties you tested contain the "positive" allele.Following
- Luisa F. Jimenez added an answer:32Alternatives to Trans-Blot Turbo PVDF packsMy lab has a Trans Blot Turbo from Bio-Rad and as far as I can tell you can only get the high speed transfers by ordering their proprietary blotting paper/buffer? I feel bad spending so much on the preassemble packages. Has anyone ever tried to get the rapid transfers on their own?
This info is very helpful. I was already suspecting the alcohol. I will be making some buffers and I will keep you all posted. Have a nice week!Following
- Samuel Coulbourn Flores added an answer:48What could be the reason protein is expressed in an uninduced E. coli culture as well?I am expressing a Leishmania protein of about 35kDa in E. coli. My problem is that I always see the same band in uninduced bacteria control lysate which is quite similar to the induced culture (1mM IPTG) and the protein does not express well (low expression level). I tried different E. coli strains such as M15, BL21, TG1, XL1 blue with pQE41 vector, and got the same. My insert starts with ATG, does start codon may interrupt protein expression? Any explanations or suggestions?
Thanks guys! Never realized social media could be so useful. To me it was all about pictures of kittens.Following
- Giel Detienne added an answer:3Any advice on single worm RT-PCR to measure heat shock response?
I have been trying to run a single worm (c.elegans) RT-PCR to measure heat shock response a couple a times so far, but all of my attempts have been unsuccessful. As you can observe in the image from the gel electrophoresis attached bellow, the DNA ladder is works fine, but the samples are not showing. Keeping in mind that I am using wild type worms in this trial, I expect to see some appearance in the column. So, this makes me wonder the following:
1. Did I fail to lyse the worm properly? For each worm sample, I used 5ul of the lysis buffer (5mM Tris pH 8.0, 0.5% Tween 20, 0.5% Triton X-100, 0.25 mM EDTA, 1mg/mL proteinase K).
2. Did I degrade RNA after isolating it? I have been as diligent as one could be, wearing gloves and all times and treating samples as soon as possible---based on the protocol.
What could be the issue with the procedure?
I talked about performing a similar single worm RNA-extraction with my supervisor who has extensive experience in worm qRT-PCR. She was quite sceptical that a single worm could deliver enough RNA for all the reactions, even with this enhanced protocol. To be honest, I never checked myself.
In the paper they mention: "A single adult worm typically contained ∼35 ng total RNA, measured using Qubit RNA HS Assay kit (Life Technologies)."
As Su mentioned above, I would measure the RNA concentration right after your extraction to pinpoint the problem. I suspect you simple don't have enough RNA to begin with.
Two things that come to mind:
1. Are you using larvae instead of (big) adults?
2. It could be helpful to increase the lysation step at 60°C. We typically incubate at this temperature for one hour for proteinase K lysis of worms (when performing a genomic DNA extraction).
Good luck, and please let us know when you have solved the problem!Following
- Alexandre Bougdour added an answer:12Can anyone help me with gel electrophoresis of total RNA?I want to check the quality of RNA on non-denaturing gel. I found this method: incubate 2 ug RNA with two volumes of denaturing buffer (50 ul formamide, 20 ul formaldehyde, 10 ul 10 X MOPS, and 2 ul ethidium bromide) denature at 70C for 3 minutes and immediately place on ice. Then run on 1.2% TBE agarose gel at 90V.
Did anyone try this method or have a good experience with alternative method?Following
- Ganesh Nawale added an answer:4Dye and RNA
I have difficulties during PAGE purification for DNA/RNA chimeric ssRNA, where I am getting slow moving dye along with my RNA even after SEP-PAK. Is there any way to get rid of it ? And will this dye affect any functional properties of RNA?
Yes glycerol option is better one.
I should definitely try this.Following
- Ghazalla M Benhusein added an answer:29Which fluorescent DNA stain is the best for comet assay?Can anyone suggest a fluorescent DNA-binding dye that gives the highest signal to background ratio in comet assay. Also can anyone suggest a good software that is suitable for yeast comets' analysis.
The best one for DNA staining is Syber green.Following
- Jasmin Sutkovic added an answer:2Any advice on denaturing urea PAGE gel electrophoresis staining?
After a successful amplification PCR I am about to visualize my AFLP fragments. Most of the protocols suggests UREA PAGE. The protocol i used (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3329804/). What would be the best way to stain the gel: Silver staining or in EtBr solution? why ?
Thanks, I will try both to compare. Best regardsFollowing
- Asa Wu added an answer:7Is it ok to select all lanes in one rectangle and draw vertical line to seperate each lane for gel image analysis using ImageJ?
For using ImageJ to analyse gel image, normally I select each lane for each rectangle/box. but some guides suggest to draw a rectangle around all the lanes of gel image in one box and then plot lane, which is easier by the way.
Is it ok to do that to summarize the data in publication?
For lane background subtraction,
which one is better to:
1) draw vertical line to cut/seperate peak from background (first figure)
2) draw line between end point of the peak (second figure)
Thank you very much
Thank you DraganaFollowing
- Thillai Punitha added an answer:9Molecular weight marker for native PAGE of basic protein?I am planing to run Native PAGE for a protein with pI around 9.3 for the purpose of checking its total weight. I am aware of the fact that I have to flip the electrodes for my protein to run in the PAGE gel (without the SDS) but how do I measure the size of the protein. I think I cannot use BSA also since its pI is 4.7.Kindly suggest any cheap molecular weight marker that I can use to determine the size of my protein.Following
- R. Jagathesh Chandra Bose added an answer:12Can coomassie stained gels which have been destained, be stained again? With coomassie stain and silver stain?I have stained my SDS-PAGE with coomassie blue but bands are very light. Probably, the stain was not good. Can the gel be stained again with new coomassie or silver stain?
Will the staining be efficient?Following
- Romanov Vladimir added an answer:97What do you use to stain SDS/PAGE protein gels?Have been looking for a fast sensitive way to stain SDS/PAGE gels, but they all required 3x washes in DI water (other than the old Methanol/acetic acid Coomassie method).
Have used the stain free gels from BioRad and they work but I have to take them to an imager outside of my lab. Then I tried AcquaStain from Bulldog Bio, a Coomassie based stain that you can put your gel right into without having to do any water washes, fast (~5 min. to see bands), sensitive, low background.
To save the results I put the gel in clear file, remove the cloth drops of water and scans on an office scanner.Following
- Robert J Meagher added an answer:3What factors or components am I missing in my LAMP assay and how important is betain-presence?I am trying to optimize loop mediated isothermal amplification (LAMP) conditions that favor a specific bacterial species. Each reaction contains FIP/BIP, F3/B3, and LF/LB primers, dNTP, amplification buffer, as well as Bst. 2.0; concentrations based on related publication. My positive control is set at 1000 CFU/reaction, has inconsistently appeared on my 1.5x gel. When I prepare a LAMP mixture for 1-10 reactions the results are strong, however, when I run 15-96 sample reactions simultaneously, the positive control is inconsistent. I tried thoroughly mixing the LAMP mixture without any positive gain. At first I thought I was exhibiting some type of human error or overall mistake but at this point I made sure to rule that out. I have even tried to denature the template DNA prior to reaction to ensure amplification, however, my results were stronger without it.
I have found betaine to be completely unnecessary in some cases. Probably more important if you have a highly structured target. Caveat, I am usually doing RT-LAMP for ssRNA targets.
Going back to very old LAMP literature, c. 2001, they showed denaturing prior to amplification was not necessary. I think at the temperature of the reaction the templates "breathe" a little bit, forming transient bubbles that allow primers a chance to anneal. And the strand displacing polymerase takes care of the rest.
That said, some primer sets are better than others - you can see 2 orders of magnitude difference in sensitivity, and a large difference in speed, depending on the primer design. It is hard to discern from software what is good and what isn't good, so it's more a matter of getting a few good candidate sets and trying them all.Following
- Chiara Benedetto added an answer:6Problems with RT-PCR (shRNAs testing)
I am testing some shRNAs by RT-PCR and I do not have hands-on experience with this technique.
In particular, I tested some primers and a PCR reaction for the very first time, but I obtained the expected signal just for the positive control (without shRNA). Before starting any experiment, I made sure that primers sequences were correct, as well as amplicon size, and I set an annealing temperature based on primers Tm.
I cannot justify my result by saying that all my shRNAs are working, because I do not have any signal in my shRNA control! What can I do to optimize my RT-PCR?
Many thanks for your answers.
actually the housekeeping gene yields the same signal in all the samples. What I did not manage to see was the signal in the "empty vector", but now I've optimized my RT-PCR and it works.
Thanks again for all your useful suggestions, they helped me a lot! And good research!Following
- Colin Germer added an answer:5Does anyone use the iBlot gel transfer system?
We are trying to perform Western blots looking for a 1KDa protein and can't seem to find any bands in that range. The smaller bands on our ladder don't seem to transfer well when we image them, would a wet transfer be more effective?
I use P3 for 7.Following
- Muhammed Burak Batır added an answer:9MicroRNAs and Gel ElectrophoresisDoes anyone have experience in gel electrophoresis with microRNA samples?
I wanted to use this method after my reverse transcription as a cheap control for my –RT reaction for every sample, to verify that I have no contamination/DNA contamination and wondered if it is possible to detect microRNAs on a gel (if there would be some contaminations)?
Mix the related matter in a erlenmeyer (bleach agarose gel) = 1 ml traditional liquid bleach (without parfume), 100 ml 1x TAE or TBE, 4 gr molecular grade agarose, 15-20 microliter redsafe, etbr or etc. . Run your gel for 20-25 minutes at the 7 Volt/cm. Analyze your miRNA or total RNA on bleach agarose gel.
- Sadia Idrees added an answer:6Could you take a look at those DNA fragmentation assay results?
The image shows DNA fragmentation assay of cells treated with toxin. I'm not sure how to read those results. For sure there is fragmentation in well with number 1, but what about wells 2 and 3? Does anyone know what this smear could mean?
Are these all black color bands are the DNA bands treated with toxin?Following
- Abdelrahim Ahmad Hunaiti added an answer:4How long can I store my sliced polyacrylamide gel fragments at -20c before proceeding with gel elution?
I have done a Differential Gradient Gel Electrophoresis using GC clamp primers and would like to know for how long the sliced polyacrylamide gel fragments will be stable at -20c prior to carrying out a kit based gel elution.
I suggest to rap your gel in aplastic cling foil and put them in 4 degree refrigerator for a week or so without a problem.Following
- Telmo Graça added an answer:14What are some common issues with SDS-PAGE that could be causing some problems our lab is facing?
Our lab has currently run into some problems involving protein migration in our SDS gels, and I was wondering if anyone has experienced something similar, or could provide some insight into what might be causing our issues.
I have attached an image for you to see.
We have suddenly had an issue where when we try to separate protein samples with SDS-PAGE, our higher molecular weight proteins merge into a single line (which is also visible as a translucent "bump" going through the gel). Oddly enough in this image, this line happens a bit lower (right above the dye front). I tried using Coomassie to see if maybe it was our ladder having issues migrating, but the protein stain also showed the protein samples were running exactly how the ladder appears.
The gel will start off running normally, with good separation of our ladder (each band is visible), but then the higher weighted bands will merge back together as a single line, and will slowly overtake every other band. Additionally, these gels have been running quite a bit more slowly than normal. The image attached to this was after about 2-3 hours at 120 volts.
So far, we have remade all of our solutions, have used another labs solutions, tried changing power packs, and have tried different cassettes incase it was faulty wiring. Nothing has been working, and we are all out of ideas.
The time that it takes to run the gel suggests that you have a much higher gel% concentration than you think or, too much resistance in the electric current conductivity (too much salts). Since you are able to load the gel I would look at the separation and not at the stacking part of the gel. I would also look at:
- Loading sample composition: As Nancy said, look at the loading sample buffer and your sample composition. Be sure you don’t have to may salts, Guanidine (precipitated with SDS and affects contiguous gels) or interfering detergents.
- Gel and Buffers percentage: Make sure that when preparing the gel you are diluting your stock solutions and not using them 10X
- Reagents: Finally, I think you should check the expire date of your reagents.
These may seem silly, but I have encountered all of them. Good luckFollowing
- Michael B LoMonaco added an answer:1For how long can I store my sliced polyacrylamide gel fragments at -20c before proceeding with gel elution following a DGGE experiment?
I have done a Denaturing Gradient Gel Electrophoresis using GC clamp primers and would like to know for how long the sliced polyacrylamide gel fragments will be stable at -20c prior to carrying out a kit based gel elution.
If your DNA fragment is buffered at -20C above about pH 7.5 it will last for months. Some depurination may occur if it is too acidFollowing
- Marc-Antoine Perrenoud added an answer:8Can I get a suggestion on the appropriate voltage and time to run agarose gel electrophoresis of bacterial PCR products?
I have noticed various authors use different voltage and time while running their gel electrophoresis such as 100V for 30 min and 75V for 35 min etc.
Can anyone kindly suggest a preferred condition so i can obtain good bands.
I am using 1% agarose with TAE buffer stained with midorin green
In our Lab we run several type of bacterial PCR product and we use 90V for 30min. Works fine for all.
Hope it helps.Following
- Subbaiah C Chalivendra added an answer:9Is it possible to use Tris-Tricine Running buffer on a Tris-Glycine precast gel?...because we have Tris-Glycine precast gels left, but the SDS-Page will be used for protein sequencing were Glycine in the running buffer is spoiling the results. So is it possible to use Tricine as a running buffer? Does anyone anyone have experience with that?
I agree with Xuezhi Bi. If you want to avoid free glycine interfering with sequencing, you can use a transfer buffer without glycine. There are any number of buffers for protein transfer when used for sequencing (e.g., CAPS, Bis-Tris or Tris-Borate). A short pre-transfer equilibration of the gel with the transfer buffer and post-transfer processing (washing, staining & destaining) should remove much of the glycine, if any, bound to the membrane or to the protein of your interest.Following
- Dmitry S Karpov added an answer:13How could I check whether there is RNA in my protein solution?
I purified a recombinant RNA binding protein from E.coli, I worried that my RNA-binding protein was contaminated with host RNA during purification. I need to remove RNA completely, I have treated with RNase A and use 1M salt in the buffer, and How can I check whether there is still RNA in the protein solution during purification? such as gel electrophoresis or NanoDrop based on 280/260? Thanks a lot.
After treatment with RNAse, the RNA fraction bound to your protein and thus protected from RNAse may be so small that could not be reliably detected spectrophotometrically at 260/280 using NanoDrop. Try much sensitive Qubit fluorometer with RNA specific fluorescent dyes. Qubit measures picograms while Nanodrop from 5 nanogramm of nucleic acids in microliter.Following
- Jane Yeadon added an answer:20Can anyone help me to find a protocol for gel extraction without using kits?I used some kits for gel extraction but I did not get a satisfying result, so I want to do it without using a kit.
Easy old-school way (though you may have to use LMT agarose and I don't know if TBE is a good idea). Cut out out the agarose strip containing the band of interest and put in small zip lock bag. Freeze at -20C. Squeeze the frozen bad through the bag with your fingers (gently) and remove the liquid with a micropipete. Clean up with phenol/choroform or EtOH precipitate if you have enough. Works for fragments of 1-4kb approx.Following
- Wolfgang Hennig added an answer:45Failed DNA sequencing reaction
I sent my PCR products for sequencing. Unfortunately all of them have noise. After post PCR gel electrophoresis I had very sharp bands without any unspecific bands or dimer primer. What could be the cause of this? What can I do with these noises to interpret the results?
I attached one sample of the results.
First, you should be able to read the sequence reasonably from your scan. I am sure that you have a second binding site with lower efficiency giving the "noise". Programs for designing primers are never a secure way, you could try to shift the sequnce slightly. I never use programs, but check for secondary structure. But this needs experience. To check whether a primer has different targets in your DNA you can use programs offering the possibilty to check each strand of your DNA with the primer (it could bind to the opposite strand too!) if you use PCR products. You might have to check your primers on the entire genomic sequence as well as you might pick up other parts of the genome by the initial PCR. The sequence seen in your scan looks actually good, so there is no impurity of your probe.
- Laurent Houzet added an answer:8How can I eliminate non-specific amplification during SYBR qPCR?
I have been designing qPCR primers for use in assays with Promega GoTaq qPCR mastermix. This uses BRYT Green, which is supposed to be comparable to SYBR chemistry. Using a non-precious positive control cDNA sample to optimise primer concentration and temperature initially, I select conditions that give single melt curve peaks/gel electrophoresis bands, blank NTCs and high efficiency. These are inducible genes with quite late Cq values. However, when I run the gene assay on my experimental samples, I get non-specific bumps/peaks in the melt curves, sometimes even in addition to the gene-specific peak in a given sample. I guess primers can act in unexpected ways in the presence of DNA with low amounts of template, that can’t be predicted from template-abundant positive control and cDNA-free reactions. I always run my RNA samples on an agarose gel so I know my experimental samples aren’t degraded.
Would anyone have any suggestions for how to tackle this? I am aware that TaqMan is probably more appropriate for my situation but we don’t have the money or time in the lab to switch at this point. I try to keep primer concentrations to 200-300nM, as I’m afraid going lower would mean I wouldn’t be able to detect anything in my experimental samples at all. I aim to reverse transcribe 1ug of RNA per cDNA synthesis reaction, though for some sets of experiments I have had to reduce this to 300ng. I dilute this 1:10 and use 2ul cDNA in a 20ul qPCR reaction volume- perhaps I should be adding more cDNA?
Any suggestions or comments on people’s own experiences welcome. Thanks!
Trizol is the best method for RNA extraction except when extracting very high numbers of samples. Of course washing step (2x, 75% EtOH, removing drops) is very important. Always better to do a DNase (liquid better, 30min, +phenolChlo, +Chlo) since DNA conta not necessary visible on a gel.
SYBRgreen is OK. For your QPCR trouble, take a non precious positive sample, dilute in a non precious negative sample (meaning RNA from cells without your target) to obtain a non precious trouble maker sample. Then optimize the QPCR conditions: if you are in a 2 steps, then go to 3 steps and try from 58 to 65C annealing T and see if you find a better condition. You can also use different QPCR mix from other companies that can give very different results especially with trouble maker primer pairs. If nothing is working, then try to redesign at least one of the primers...Following
About Gel Electrophoresis
Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.