When I was performing a PCR for some genes and running the PCR products on the agarose gel, I observed the presence of tailing and the bands were strong. Do you think I needed to change my PCR conditions or the gel concentration?
Thank you very much.
If your bands are very strong, then the gel is probably overloaded. With too much DNA in a lane, there can be a smear above the band because it does not migrate properly. The tailing will likely disappear if you load less PCR product in each well.
Alternatively, you could reduce the number of cycles in the PCR. Each cycle you eliminate cuts the amount of product in half.
Including all the above mentioned solutions also clean the combs nicely before running the gel..I think all the solutions mention should solve your problem....
As several people have noted above, you need to find the right run conditions, which will vary depending on the size of the product that you are interested in. Things you can optimize include:
-the percentage of agarose in your gel (see http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html for guidelines),
-the amount of salt in your sample (this should only be a problem if you are concentrating samples with salts in them),
-the amount of DNA in the sample (too much DNA will overload the gel and cause poor separation; maximum amount of DNA in a clear, well-defined band is usually around 100ng),
-the thickness of the gel (optimally 3-4mm; thicker gels can result in fuzzier bands),
-the type of agarose you use (for certain size ranges, different types of agarose can provide clearer resolution),
-the voltage (running too slow allows the DNA to diffuse, and too fast causes poor separation; aim for 4-10 V/cm as measured between the anode and cathode),
-amount of buffer (the gel should be submerged in buffer, but I've heard that having too much buffer on top of the gel can cause problems; not sure if this is true),
-and importantly, how you are staining your gel. Including your stain in the gel while the gel is running can change migration patterns. Consider trying a post-staining method.
I found this website to be particularly informative:
https://www.lifetechnologies.com/us/en/home/life-science/pcr/elevate-pcr-research/agarose-content-with-tips-and-tricks.html
Maybe we should back up a bit and ask what you're hoping to do with this gel/PCR product. Do you just want to confirm that it amplified? Are you planning to do a gel extraction? Those lanes look fine to me, really...
Hi
The gel looking like It has genomic DNA contamination(just close to the well).
So please reduce the template (DNA) in your PCR step.
The bands are look fine so you can get nice amplification even with low template, so just serial dilute your DNA and do PCR with all the diluted DNA you can get clear band without tailing.
Good luck
Yes, Anandhakumar, I agree with you that the samples are contaminated with genomic DNA (exept for the sample 6) and in some samples genomic DNA band is quite bright, suggesting rather high amount of template. Mohamed, my advice is also to considerably reduce the concentration of the DNA template and to equalize them in all the samples before running PCR.
I don't think you should call something a contaminant when you added it to the reaction and never attempted to remove it. The template used for your PCR reaction will be present in the gel unless you've somehow done a size-selection to remove it. Yes, decreasing the amount of template in the PCR reaction will decrease the visibility of the genomic DNA band, but the DNA will still be there-- and there is no need to worry about that. If you need a PCR product that excludes everything except your target, just do a gel-selection.
You're right, Jeremy, the contamination is too strong term; I didn't think about it. However, Mohamed didn't mention what is he planning to do with the PCR product, so I'm just focusing here on his question about tailing reasons and how to avoid it.
hi,
1.Use low range marker so that u can get clear band
2. reduce the gel % so that high moloecular weight may retain in the well itself and the clarity may be fine.
3.The angle u taken the gel photo slightly various it seems to me.
4.If tailing occurs means lane in between wont be correct. so no tailing
5.the bands 1,2,4,6,7 are good 3&5 are lighter
6.Ofcourse contamination is there u ultra centrifuge the samle and then amplify
7.increase your runing for time
gud luk
Thank you for all your valuable answers and advices
Really I'm so happy for that
Best wishes and thnx
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