- Harlan R Barker added an answer:Is it possible to construct statistically a phylogenetic tree based on Relative mobility values calculated from enzyme gel electrophoresis?
for ex if Rm values are 1. 0.3, 0.6,
2. 0.4, 0.9
3. 0.35, 0.65
These values are not sufficient for constructing a phylogenetic tree. Your best presentation of this data would be as a graph where similar mobility values are grouped together.Following
- Ales Kovarik added an answer:Can anyone explain the secondary DNA bands in agarose gel (besides a-specific amplification in PCR)?In some of my qPCR assays 'secondary bands' or 'ghost bands' seem to appear, which runs shortly after my expected band, when I run the products on an agarose-gel electrophoresis.
Now, I have fairly extensive experience with PCR and gel electrophoresis, and I understand that the easiest explanation is that a second product is actually being amplified in my PCR. However, I don't believe this is the case. A few reasons why I think this are:
-The melting curve shows one very neat and distinct peak. I realise that this is not proof that no secondary product with the same Tm is present.
-several PCR assays seem to have this problem for me, and the secondary band always seems to run just a bit slower than the expected band.
-Negative samples never show a band at the height of the 'secondary band'. So if it would be another product, it never seems to be amplified from samples which are negative for my initial target.
-When I sequence the PCR product I get nice and clean sequence data without ambiguities or high background noise, what you would expect since the 'secondary band' seems to be high enough in concentration to cause these.
I extracted both bands from one of my gels, and I'm currently cloning them both (seperatly ofcourse) into a pGEM T-EASY vector, so that I can check the sequence of these inserts independently of the PCR primers used. I want to check if the sequences are actually the same or not, and thus if I'm dealing with the same product or not.
Could this be some sort of secondary structure in which the product is migrating through my gel? It's driving me crazy and I would love to hear if anyone else has any experience and hopefully an explanation for this.
See attached one of the gels. The product should be 149 bp, which seems to correspond with the lower band (fastest migrating). It's a 100 bp marker.
As mentioned, I cloned both fragments which I first extracted independently from one of my gels. I have now sequenced the inserts and the sequence appeared to be identical.
Thanks for the comment, Nicola. Yes, you are right. DNA binding proteins certainly influence DNA mobility in gels. However, Taq polymerase is an unlikely candidate since it dissociates rapidly from double stranded DNA after the polymerisaiton.. Best. ALesFollowing
- Guetouache Mourad added an answer:How long does it take to form 8.5% acrylamide gel with 50ml volume for DGGE?
I use acrylamide gel with 8.5% concentration with 50ml volume but I think it takes too long waiting them forming unto gel, about more than 8 hours, even overnight. In contrast, my friend also use the same gel concentration with 10ml volume and it only takes 10 minutes until it becomes gel. I wonder why, because I need the gel to form quickly.
DGGE was performed using the protocol of Muyzer et al. (1993). Gradients ranging from 35% to 70% of denaturing agents were used for separation of both PCR products from archaeal primer pair A934f/A1390r-GC and bacterial primers 984f-GC/1378r, respectively. Gradients were poured with 7% [w/v] acrylamide in the low denaturing solution and 8% acrylamide in the higher concentrated one; 100% denaturant corresponds to 7 M urea and 40% [v/v] formamide. Electrophoresis was run at a voltage of 100 V in 1× TAE buffer (Tris–acetate–EDTA) for 16 h at a constant temperature of 60° using the Ingeny phorU mutation detection system. Gels were silver stained (Radojkovic and Kusic, 2000) and conserved in preservation foil. Equal amounts of DNA, verified by PicoGreen measurement, were loaded onto the denaturing gels to allow a densitometric comparison of band patterns. Band patterns were compared and checked on cluster formation (Pearson correlation coefficient, UPGMA) using GelCompar II software (Applied Maths) (Cornelia and Paul, 2008).Following
- John LaCava added an answer:What is difference between TAE and TBE buffers and their properties regarding use in agarose gel electrophoresis?Why do some researchers use TAE and some TBE?
Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis
Brian A. Sanderson, Naoko Araki, Jennifer L. Lilley, Gilberto Guerrero, L. Kevin Lewis
0.5x TBE working very well for me.Following
- Sirisha Aluri added an answer:Can Agilent Bioanalyzer detect genomic DNA contamination for RNA samples? Is the data (RIN value) generated by Bioanalyzer relevant?I have recently extracted total RNA from a plant. Upon extracting, I QC checked the RNA via NanoDrop, gel electrophoresis and Bioanalyzer. The NanoDrop reading showed good RNA concentration as well as good A260/280 values (ranging from 1.8-2.10) with acceptable A260/230 absorbance for some samples (2.0-210). I then electrophrosed the same RNA samples on agarose gel to check its integrity. The agarose results showed that the RNA was contaminated with genomic DNA and that it was degraded to a certain level. To verify the results, I then proceeded to check these samples via the Agilent Bioanalyzer. However, the results generated by the bioanalyzer contradicted to the results produced by agarose gel. The bioanalyzer showed that the extracted RNA samples were free from any form of genomic DNA contamination. Also, the RIN value for some of the RNA samples was above 8.0. Additionally, no any additional peak (Image not shown) neither a distinct band corresponding to the gDNA contamination was observed when the samples were run on the Bioanalyzer
My concern is, is the RIN value generated by the bioanalyzer reliable? Also, why did the bionalyzer not show any traces of gDNA contamination? Which data is much more reliable (Bioanalyzer or Agarose)?
I have attached images of agarose and the bands generated by Agilent Bioanalyzer for your convenience. Kindly take a look at them.
The gel, gel cast and the flask used to prepare the agarose gel were all treated with RNase Away. Also, the TAE buffer was prepared by using Milli-Q water (filtered, RNase free)
Thank you in advance.
Check for DNA contamination using Qubit dsDNA kit. It´s specific, quick and easy. Sorry for much delayed post but may be someone else can be profited.Following
- Vanessa Mendes added an answer:What are the causes of a bad Western blot result?
I did the Ponceau on the last run, and it came out looking like these two images, attached.
What could cause this, besides bad gel?
I'm having the same problem. How did you solve it? Today I tried more sonicating and DNAse I, but it didn't seem to improve.
- Christian Q. Scheckhuber added an answer:Any help with Blue Native PAGE as my protein is not running into gel?
I am running a Blue Native PAGE (BN-PAGE) and I have been running the gel for 2 hours at 40 V at 4°C and my samples are not running into the gel. The markers are not running into the gel either, so I know it's not just an issue with the charge/amount of protein loaded. I'm trying to isolate a complex that is about 500 kDa. I did not boil my samples or anything, I simply dissolved them in a small amount of detergent (0.5% or 2.5% DDM, or 1% SDS), incubated for an hour on a rocker at 4°C, pelleted out insoluble material, added coomassie brilliant blue G250 to 25% of that of the amount of detergent (from a 5% solution in DI water), and loaded them on the gel. I developed my protocol from Schagger et al., 1993.
Here's some info on my protocol:
Gel: 4-12% gradient gel (ExpressPlus PAGE Gel; GenScript M41210)
Sample Buffer: 50 mM bis-Tris, 50 mM NaCl, 1 mM EDTA, 5 mM 6-aminocaproic acid, 10% glycerol, pH 7.2
Anode Running Buffer: 50 mM bis-Tris, 50 mM Tricine, pH 6.8
Cathode Running Buffer (First 1/3): 50 mM bis-Tris, 50 mM Tricine, 0.02% Coomassie brilliant blue (CBB) G250, pH 6.8
Light Blue Cathode Buffer: 50 mM bis-Tris, 50 mM Tricine, 0.002% CBB G250, pH 6.8
Molecular Weight Markers: Native Mark (Invitrogen)
Nice to hear that it is working now!
Best regards, ChristianFollowing
- Lenin Rueda added an answer:Which 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.
- Zandisiwe Magwebu added an answer:Failed 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.
I've designed the primers using Primer3 and NCBI primer blast. We always start with gradient PCR (40-65) and choose the best temperature. The PCR product was clean, did not show primer dimers. However, the primers that i've designed with primer3 had dimers and i'm still optimizing with no luck. DNA was quantified immediately after extraction and it was indeed clean. I will try to use the nested primers next time and see if my problem will be resolved. Can you kindly recommend a tool that I can use to check if my primers have additional binding site? Thank you so much for your feedback.Following
- Tim Hunt added an answer:Can 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?
I just ran NEB 1kb DNA Ladder and NEB ssRNA Ladder on a 1.2% TAE gel following denaturing as per attached paper Denaturing RNA Electrophoresis in TAE gels (2005). Adding 60% Formamide, 1µg Ethidium Bromide, and loading dye and heating @ 65˚C for 5 min, followed by Ice bath and directly loading in to wells. Ladders were electrophoresed @ 50v for 1.5 hours. Lane one shows 1kb ladder placed in gel without treatment, Lane 2 and 3 show to 1kb DNA ladder following denaturing conditions and ssRNA ladder. Notice the 3kb matches well, but there is a decent amount of drift in the 9kb region and a small amount at the 1kb region (500 is at very bottom of image lane 2 -very hard to see), but for estimates it might be sufficient.Following
- Anouk Spelt added an answer:I am having a problem with distinguishing two bands in the gel electroforese, can anyone help??
I'm attempting to determine the sex of African penguins (both parents and chicks). I extracted the DNA from feather samples (Chelex protocol) and now using the primer pair 2550F and 2718R (Fridolfsson). I used a the same PCR protocol (Fridolfsson) but with a gradient to see which annealing temperature is the best (50-60 C). Attached the photo of my gel (1.5% TAE, 75 Volt, 30 min). On the top 8 gradient wells is DNA from a male adult placed and top right 8 wells from a female adult (bottom are unknown chicks).
It seems that the annealing temperature does not make a difference, does anyone has a suggestion how to improve the bands on the gel? Furthermore, the first band (600 bp) is more intense than the second band (450 bp). Females should show two bands (450 + 600) whereas male should show only one band (600 bp). On this gel I see several bands (also in some samples of the male).
Does someone have a suggestion to improve this analysis and/or the reason why the second band is less intense?
Thank you Matthew and Gizella :).
I hadn't the possibility to measure the DNA concentration due to labconditions, but it worked out in the end. In the futer I will try the vertical gel system you suggested Gizella, thank you.Following
- Charumathi Jayachandran added an answer:During silver staining I am unable to stain the gel - it remains blank as if it were freshly cast. How can I overcome this problem?I have isolated a protein from cell lines and I want to confirm the presence of the protein by Bradford reagent. But when trying to silver stain, I am unable to stain the gel - it remains blank like it was freshly cast. I have followed the standard protocol of 0.02% Sodium thiosulfate for one min 0.2% silver nitrate. I used a standard developer and a fixative. When discarding the reagents, the colour of the waste turns a black/brown colour. How can I overcome this problem?Following
- David Farringdon Spencer added an answer:What is the role of Sodium Cacodylate in a DNA buffer?
I am doing an electrophoretic mobility study on plasmid DNA (pBR322) treated with cisplatin.
The paper I am following says I have to dilute my DNA in a 1mM sodium cacodylate and 20mM NaCl buffer.
I understand the purpose of the NaCl, but not the sodium cacodylate. The literature I have so far come across tells me that this compound is often used for the preparation of microscopy samples, as it prevents the mitochondria and other organelles from rupturing. However, I was unable to find out what the purpose of this compound could be in an experiment involving free DNA (not cells).
They also use xylene (4mL!) to quench the reaction after incubation (I presume the reaction of cisplatin with DNA), and I am not sure why they do that either. Thought I would mention it, maybe it has something to do with the sodium cacodylate...
I would be very grateful if someone could point me in the right direction.
Thanks in advance!
- Ivan Brukner added an answer:What is the best protocol for 16sr RNA gene PCR?
I have problem when I want to PCR 16sr RNA gene of my unknown strain. I have extracted its DNA and did PCR by using lyophilized master mix tube for PCR. The result was good but I had an extra band in my gel electrophoresis. I did the PCR again but the problems exist. So I extracted the DNA by using another Kit and did PCR with another PCR pack. But the result was not good. I think my problem is in mastermix preparation or in my thermal program. Can anyone give me an advice to solve this problem?
Assays trying to measure total bacterial load are usually based on the amplification of universal segments of 16S rRNA genes. Previous assays were not adoptable to “direct” PCR protocols and/or they were not compatible with hydrolysis-based detection. Using the latest summary of universal 16S sequence motifs present in literature and testing our design with 500 liquid and 50 formed stool samples, we illustrate the performance characteristics of a new 16S qPCR assay which addresses well-known technical problems, including (a) positive priming reaction in the absence of intended target due to self-priming and/or mis-priming of unintended targets; (b) amplification bias due to non-optimal primer/probe coverage; and (c) too large amplicons for clinical qPCR. Stool swabs ranked into bins of different bacterial loads, show significant correlation with Ct values of our new assay. To best of our knowledge, this is the first description of qPCR assay measuring individual differences of total bacterial load present in human stool.Following
- Kate Karelina added an answer:Why aren't my samples stacking during SDS-PAGE?
I've been having a problem with my samples not stacking well during SDS-PAGE. I'm following a protocol that has worked very well for the past year or so and is for some reason having issues now. I use bio-rad precast gels (#456-1086), I load 40ug sample and run at 80V, then increase to 120V. I have checked the buffer pH and make it fresh each time. I've tried buying new gels. The attached picture is representative of what it looks like, the samples run that way all the way down the gel, they never stack properly. I appreciate any suggestions!
Update: thank you all again for your advice. I narrowed the problem down to the running buffer, it was being made with Tris HCl and pH adjusted. Switching to Tris Base has completely fixed the problem.Following
- Ranu Pal added an answer:Dark bands on edges of Western Blot and faint development of bands in the center of WB, even in the loading control?I am trying to replicate an experiment previously conducted, and observed that irrespective of the samples loaded, on developing the WB by chemiluminescent substrate, the bands on both edges of the blot appear darker than the bands in center. So on looking from left to right dark bands slowly fade and become lighter, before becoming dark again on reaching the right side of the WB. This is particularly troublesome since this is observed even in case of the loading control (GAPDH).
I agree with most of the answers. There are few suggestions on this topic.
1. If it is pre-cast gel, check the date of expiry.
2. After placing everything for sandwich, run a 15ml tube across the sandich before clamping to make sure there is no air-bubble trapped.
3. Place a container full of ice inside the transfer tank so the chamber does not get too hot.
4. Incubate the blots in a container that flat surface so the blots get covered with the buffer containing either primary and/or secondary antibody.
5. Do not let your blots dry completely during chemiluminescence procedure.
Hope it helps.Following
- Guy Bouvier added an answer:What is the best way to degrease glass slides for the Comet Assay?
I have tried ethanol and methanol, and the agarose still ends up at the center of the slides before it is completely dried or it does not cover the periphery of the slides. Any suggestions? I am having a hard time with this one. Thanks.
burn the slides (both sides) to get rid of any residual material, it is the easiest and quickest way I have used.Following
- Kumar Sharad added an answer:Why does my prestained protein marker disappear at the end of the SDS-PG run?I have a problem with my SDS-page gel. In the beginning of the running the marker bands look fine but disappear later. The separating and stacking gels were 15% and 4%, respectively. All the buffers were freshly prepared. The bands from my sample were diffused to the next lane too. All the lanes were occupied (contained different samples). I repeated this twice and with each time all the buffer is freshly prepared and pH is adjusted.
Can anyone help to explain what is the problem?
Yes I have also same problem .I have prepare freshly buffer 2-3 time and adjust the pH but prestained protein marker disappear at the end of the SDS-PG run? I have used prestrained laddaer in diiferent company but same problem occurs.
Can anyone help to explain what is the problem .Following
- Pranay Amruth Maroju added an answer:Is anybody working with DNA methylation using MSAP?
I faced problem to stain and visualize denaturing PAGE gel. I will appreciate if you share your practical experience in this issue.
I hope you are following post run staining protocol where you remove your PAGE gel from cassette and release it into a 100ml TAE buffer containing 7-10ul EtBr and put it on a rocker for 10 min and then wash it with 100ml of TAE buffer without EtBr and then look on a UV Transilluminator. This method works fine.Following
- Lori M. Kelman added an answer:Can agar be used instead of agarose to run an electrophoresis gel?
Can agar be used instead of agarose to run an electrophoresis gel? Or will the bands not be defined enough?
Just in case you are asking if you can do something cheaper than agarose as a demonstration for school children, in that case you can use almost anything, including lemon Jell-O, for a demonstration of gel electrophoresis. But NOT for research.Following
- Chongxu Zhang added an answer:I am having an issue with my samples pooling when running a Western for a 150 kDa protein. Any tips?
I am not sure what is causing the pooling so if anyone has some troubleshooting tips please let me know. We are running on 4-12% gradient bis-tris gels using NuPage SDS buffers. We do denaturing gel electrophoresis and load 10 ug of protein. I currently transfer onto nitrocellulose but I understand that PVDF may work better for larger proteins. Thanks for your help!
Try to load 30ug total protein.Following
- Annarita Oranger added an answer:What does increasing both LC3 II and p62 mean?
recently, I tried to check the role of one kinase (called "A") in autophagy. I treated cancer cells with A inhibitor (KI), in which A is oncogenic kinase for driving cell proliferation. However I found both LC3 II and p62 expression were increased (see attached figure). So I got confused. I expected A could suppress autophagy. Does anyone have any idea for the case. Furthermore, I also treated the cells with autophagy activator ABT737 (Bcl2 inhibitor). Compared to both untreated cells and starved cells, ABT737 treatment caused less LC3 II and p62 expression. Why the tendency of LC3 II and p62 is similar? Thank you for your attention.
I suggest you this extraordinary article in which is well explained how to interpret p62 western blot assay and every kind of aspect in monitoring autophagy:
p62 during high autophagic flux is up-regulated both at transcriptional and proteic levels so, even though generally autophagic process causes p62 decrease, up-regulation of p62 expression could cause p62 increase in WB and sometimes no variations in p62 blots compared to control sample.Following
- Henk-jan Schoonbeek added an answer:Would 97% vs 99% guandine thiocyanate have an effect in gel extraction buffer?
I am interested in making my own Qiagen buffers and the 97% is less than half the price of the 99%
Yeah give it a go, and try what Philuppe saidFollowing
- Lauren Bass added an answer:I am performing pfge analysis on staphylococcus aureus strains but I am struggling to understand the results, I was wondering if anyone could help?
In the gel dated 11/12/12, what would be the reason for such an unclear result? Too much DNA, undigested DNA?
In regards to the gel dated 3/12/12, could it be said that stains 5 and 6 have the same restriction profile? Should I re run and try obtain clearer bands? Could it be said that 7 and 10 have the same restriction profile, although there is a clear 1 band difference?
I've kind off been chucked in the deep end in terms of analysing these results and I'm getting rather overwhelmed.
The DNA was cut with SmaI and a lambda ladder was used. Any help would be much appreciated.
Thank you all for your replies, I have attached the protocol that I have been using. So yes I think too much DNA and not incubated for long enough with the digestion enzyme.
I have going to have a look at all the methods posted and re run the whole procedure on Monday and hopefully get better results,
- Yuan-Yeu Yau added an answer:What is the best way to avoid smears in the gel after T7 endonuclease assay?
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
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.
1. I am not sure that the unspecific bands will affect the T7 and cause the smears. T7 endonuclease I only cut the unmatched bases of a DNA molecule (see attached figure).
2. Since your DNA sample contains unspecific bands, you should gel-extract and purify the right band first, then use it for T7 treatment. Please see this useful website ( http://www.crisprflydesign.org/t7-endo-i-assay/ ) for step-by-step protocol of T7 assay. They also suggest cut out the right band.
3. They used 10U of T7 endonuclease I, how much U did you use?
4. If you want to search a way to produce a clean single band, you can try to use a higher annealing temperature, but this depends on what are the Tm of your 2 primers. You don't want to over-shoot it.Following
- Murugadas A added an answer:DNA Agarose gel electrophoresis trouble, incomplete strange bands, any suggestions?
Hello dear friends!
Well, In our Lab we have a little issue with the agarose gel electrophoresis. When we run a PCR product we saw few times a strange band, in fact sometimes we had the strange band in our ladder (strange thing because is the same ladder we usually use for more than 6 months by now) not just in the PCR product.
We're using the same SBx1 buffer, made with the same SBx20 buffer, same quantity of agarose, electrophoresis camera, power supply, BrEd, and some tests with GelRed and we have the same issue.
The funny thing is that just some times we have this issue, because we run at day like 3-5 pcr gels but just in some of them we have this issue.
Some suggestions about what its going on here?
Images: TrnlF1 7-03-15 P.cembroides var.bicolor.JPG image is the rare one, the 03.20.15-PruebaCajas.JPG is a sample because we think was a electrophoresis camera, so we perform a test, but in both cameras worked fine, and the 21feb2015 image is a GelRed test of a PCR product, but its just fine too.
Thank you for your time :D
I can say that there may be a problem in EtBr stain and as said by Lawrence change the buffer. Added to it your DNA concentration may be high which gives smear in the gel. Try to quantify it.
- Nathan Kieswetter added an answer:What reasons might there be for contamination in a negative control template in a gel electrophoresis?
I saw three bands around base pairs of 1500, 1200, and 700 in the G-NTC for a column with DNA extracted from a human. Any ideas for the reasons of said contamination would really help.
Don't throw out all your reagents. If you've got the time, run them individually on a gel in a 'process of elimination' to see where the contamination is contained. Once discovered you can save yourself and others a lot of time and money.I agree with Mostafa above, filtered tips are essential for an PCR experimentation. Further in our lab we wipe down our pipettes and surfaces with bleach followed by 70% ethanol followed by a 15 min UV sterilization. Using new tips and working at all times in a laminar flow will further ensure protection against possible contamination.
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.