University of Oregon
Question
Asked 14 February 2020
Why is my DNA stuck in my well of an agarose gel?
HI there,
I need some assistance in figuring out why my DNA will not run down my agarose gel. Here is my experimental pipeline
1: Ligate a PCR product with custom adapters
2: Run Ligation product on a 4% agarose gel
3: Gel extract Ligated product (~160bp) (Qiagen minelute gel extraction kit)
4: Reamplify product using primers that bind adapter region
After conducting these experiments, I run the product on an agarose gel and my DNA product does not run through the gel, however my ladder does. I have performed this experiment in the past with no deviations and I was able to get my expected band sizes (See attached image). It can't be genomic DNA because my template for my step 4 is gel extracted DNA at 160bp. I've tried PCR purifying my PCR product, treating it with SDS to no avail.
Can anyone offer any suggestions? Please see the attached pictures.
Feb12 shows the ligation of my molecule and which bands I extracted
Feb14 shows the amplification of those extracted molecules
Jan 24 shows my successful attempt and what bands I received
My ladder used in all experiments is generuler 50bp and in most reactions I loaded ~400-600ng of DNA.
Would appreciate help in figuring this out!
Thanks!
Most recent answer
This happens when the DNA size is small and it is probable that it primes itself. I had the same problem and it resolved by increasing the primer concentration in PCR.
Popular answers (1)
University of Zanjan
It happens due to one of these reasons;
1-Poorly-formed gel wells:
Remove the gel comb only after complete polymerization of the gel. Pour the buffer onto the gel immediately. Rinse the wells with electrophoresis buffer to remove urea from denaturing polyacrylamide gels prior to loading the sample
2-Excess DNA loaded:
Follow the recommendations for loading described in the certificate of analysis of the DNA ladders/markers (~0.1-0.2 µg per 1mm gel lane width) or in the Table 9.6 on p.445. If possible load the same quantity of the sample.
3- contamination of the DNA sample
4- gel shift affect:
The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes may alter DNA migration in the gel and cause the DNA to remain in the gel wells. Lambda DNA or other DNA with long complementary overhangs may anneal resulting in an atypical band migration pattern. To eliminate these effects, use 6X DNA Loading Dye & SDS Solution which is supplemented with 1% SDS to eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends. Always heat these samples with SDS at 65°C for 10 min, chill on ice, spin down and load.
7 Recommendations
All Answers (28)
University of Cologne
Maybe while pipetting you accidentally pierced the bottom of the well with the tip and pipetted the samples slightly under the gel?
1 Recommendation
Fusion Pharmaceuticals
This has been a result i've been getting 3x in a row. If it happened once then possibly but its not a pipetting error
University College London
Is it possible that you are over amplifying. 20 cycles is 1000000 times more pcr product than the original amount and then primer is not in huge excess and product may self bind in ever longer products which can amplify and further concatenate to ever longer products. Try diluting the initial product 1/100 and run further amplifications at 10,12.....20 cycles removing a tube at each cycle number and you may find a tamplate amount and cycle number that gives good strong amplification without generating very long concatemers
6 Recommendations
I have never had the need to make more than a 2% agarose gel, 4% seems problematic. Also 400-600 ng per well seems excessive, 100 ng or less is usually recommended per well.
Fusion Pharmaceuticals
I don't think thats the issue Paul but I can try that since its something I havent tried before, below 25 cycles. My successful attempt on Jan 24 used 35 cycles. But I repeated this Feb 14 experiment with 35 and 25 cycles and both blots demonstrated that aggregation of DNA in the wells, however, the 25 cycles did show less pronounced aggregations (probably due to less DNA being amplified). I didn't think over added template, considering I only put in 10ng of starting template.
1 Recommendation
Fusion Pharmaceuticals
Hi Amy,
I've run a 1ug on a 2% gel no problem. I ran more DNA because I am expecting multiple band sizes for the PCR amplification. 100ng is easily detected on a gel but 100ng spread over many bands won't show up. For reference, the DNA ladder is ~500ng total and it runs fine.
I get multiple bands all the time on 100 ng per well samples. For 4% gels I am seeing that people use a special "low melting" agarose product
Your setup isn't standard, and you are running too many cycles as Paul mentioned. That much DNA per well can affect how the wells beside it run.
1 Recommendation
Fusion Pharmaceuticals
Hmmm. Ok, I will reattempt two new PCRs. One where I dilute my template 1:10 and another where I run the PCR with 15cycles. I don't think that is the problem so I welcome more suggestions if they come up.
Agarose is too expensive to be running 4% gels ;-) even the microwaving of a 2% gel is troublesome, to get it all dissolved and not boil the water away, making the TAE too concentrated
University College London
10ng amplified with even 20 cycles would try to amplify to 10mg of product...not possible of course so something has to fail badly and in this case I suspect the product quality is where the failure is seen most clearly
1 Recommendation
University College London
Another aspect I missed earlier is that the copy number of target molecules in 10ng in this instance is huge compared with , for example, 10ng of genomic dna which only has 2 copies typically every 3billion bases which might also suggest over amplification
1 Recommendation
Fusion Pharmaceuticals
No not yet. i have a few things ill try starting tuesday. Will provide updates
1 Recommendation
University of Edinburgh
My guess is that your PCR conditions are producing large amounts of single strand DNA. A colleague had this problem recently - solved by tinkering with primers, reducing the number of cycles and finding the magic additive (in this case betaine rather than DMSO) to keep the PCRs producing properly annealed dsDNA instead of a mat of knotted up single strand. She satisified herself it was ssDNA by testing a stain that was more specific for single strand nucleic acid.
1 Recommendation
University of Zanjan
It happens due to one of these reasons;
1-Poorly-formed gel wells:
Remove the gel comb only after complete polymerization of the gel. Pour the buffer onto the gel immediately. Rinse the wells with electrophoresis buffer to remove urea from denaturing polyacrylamide gels prior to loading the sample
2-Excess DNA loaded:
Follow the recommendations for loading described in the certificate of analysis of the DNA ladders/markers (~0.1-0.2 µg per 1mm gel lane width) or in the Table 9.6 on p.445. If possible load the same quantity of the sample.
3- contamination of the DNA sample
4- gel shift affect:
The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes may alter DNA migration in the gel and cause the DNA to remain in the gel wells. Lambda DNA or other DNA with long complementary overhangs may anneal resulting in an atypical band migration pattern. To eliminate these effects, use 6X DNA Loading Dye & SDS Solution which is supplemented with 1% SDS to eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends. Always heat these samples with SDS at 65°C for 10 min, chill on ice, spin down and load.
7 Recommendations
alternate pcr enhancers to dmso and betaine?
The Strategy:
In order to examine their ability to enhance difficult PCR templates, the authors randomly selected 104 GC-rich human genomic amplicons with lengths between 700-800 bp and with GC content between 60-80%. The PCR additives were used at final concentrations of 1.075M for ethylene glycol and 0.816M for 1,2-propanediol. Betaine was used at a final concentration of 2.2M. All amplification reactions were performed at least 3 times to ensure the accuracy of results.
The Outcome:
The researchers put together an extensive table that summarizes the results of PCR for 104 amplicons. In summary, 13% (14) of the amplicons amplified without any PCR additives. 72% (75) worked with just betaine alone while 90% (94) worked with only 1,2-propanediol and 87% (91) were successful with the addition of ethylene glycol alone. While three reactions were rescued with betaine and not the other additives, overall performance was improved using these the ethlyene glycol or 1,2-propanediol.
Unexpected results:
Interestingly, in some cases betaine, the grand old daddy of PCR-addtives, showed a PCR inhibitive effect. Several of the reactions that worked only with ethylene glycol or only with 1,2-propanediol failed when betaine was added back into the reaction in addition to the new additive.
1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer
Helena Horáková, Iva Polakovičová, Gouse M Shaik, Jiří Eitler, Viktor Bugajev, Lubica Dráberová & Petr Dráber
BMC Biotechnology volume 11, Article number: 41 (2011) Cite this article
1 Recommendation
Florida State University
For visualization of gDNA best concentration of agarose is between 0.8-1.0 %. Decrease the concentration of your agarose gel.
2 Recommendations
Fusion Pharmaceuticals
Charles, I am not even using genomic DNA in my PCR's. That can't be the issue. I used higher % gels to resolve smaller DNA products of around 50-400bp
Fusion Pharmaceuticals
Hi everyone,
So I took everyones ideas into consideration. I did 0.5ng inputs of template reaction into my PCR, Ran 15-20 cycles, lower percentage gels. All came back with smears.
I was able to locate the source of my smears to my Qiagen gel extraction kit. I tried to run my gel extracted product on my gel and it floated away. Used Qiagen kits million times before but never had this happen. I believe my smearing was due to ethanol and salt contamination in my PCR. I ran the qiagen kit with extra wash steps and centrifugation steps to rid the extraction of Salts and ethanol and the smear still occured. I compared it to a 6 year old zymogen kit and it worked perfectly fine.
Who knew...?!
2 Recommendations
Fusion Pharmaceuticals
Paul Digard What stain was that?
I just use the freeze and squeeze method for gel extraction, requires only a filter pipette, works well. A 2% agarose gel will resolve down to 50 bp. Over and over, many of my pcr and gel problems are resolved by extra cleaning steps and dilution of template dna. When a protocol says to use pure, undiluted PCR reaction - I clean and dilute first. And in my experience, there are only problems and no significant yield gain running any pcr over 21 cycles. Too many cycles leads to primer dimer and junk concatenation product.
1 Recommendation
University of Edinburgh
Christopher - not entirely sure - some Qubit RNA stain I think - gave relatively more staining than however the machine visualises things when looking for DNA. Sorry to be vague - never used the equipment myself, I'm from an EtBr and agarose era. Just going by what was presented in a lab meeting.
I will share my experience. So i used qiagen gel extraction kit too. But have you used the reagents properly ? According to volume? Also after using isopropanol dry spin it at full for 3-4 mins. It helps to remove extra residual alcohol which interfers with the yeild..
Other wise you can chk ur gel properly. 1% agarose gels are best..
Hope my suggestions are of help. Thanks!!
Hiroshima University
I recommend the use of polyacrylamide gel electrophoresis to show the clear results for short DNA analysis, and rinse the wells with electrophoresis buffer to remove from denaturing polyacrylamide gels prior to loading the sample as Dr. Sepideh Zaeri had answered.
The ladder of electorophoresis of PCR results is leaded to the unspecific annealing of primer, and there are several way to reduce the unspecific annealing, for example to make the Mg concentration higher, or SDS concentration higher, or change the primers.
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