After the PCR step I get a light band around 400-500 bp which is the size I selected but I also get a smirr between 200-400 bp. I tried to change the annealing temperature but it doesn't change anything. I'm not sure if the problem is my PCR conditions or the ligation step.
Does anybody have an idea about which step needs to be optimized?
I'm attaching a picture of my gel (ladder: 100-700 bp with a band every 100 bp).
hi,
what do you mean by “but it doesn't change anything”？I think the most important factor which influence RAD library is restriction enzymes you choose, giving a specific species.
Hi Ying Huang,
Sorry, I should have given more details.
The six wells on my gel are occupied by PCR products (after ligation) from digestions of the same species DNA with six different pairs of enzymes. So regardless of the enzymes used the results are not what we would expect for a successful library. I checked the digestions with a BioAnalyzer beforehand.
What I meant by "it doesn't change anything" is: when I change the PCR conditions the results are similar (the gels looked the same).
Thanks for your help.
Hi Marie,
if you have selected the DNA fragments with length ranges from 400~ 500bp, the smirr is difficult to be explained. May I guess that there maybe something wrong with the method by which you select the digested DNA fragments?
What size selection method are you using, and what polymerase for PCR? In my lab we size-select on 2% agarose gel with a razor blade, which is certainly the cheap way to go (and causes some minor variation from library to library, depending on precisely where I cut) but I like that I can see exactly what I am getting. Then we use Kapa Hi-Fi (or in the past, NEB Phusion) to amplify. Maybe (a big maybe) ordinary Taq could cause the problem you see by preferentially amplifying smaller fragments. The only other thing I can think of is that you have RNA contamination, which is not a problem in and of itself, but still you should be seeing a lot more of your amplified library than degraded RNA. Our protocol can be found at: http://openwetware.org/wiki/Sacks:RAD-seq
Hi Ying Huang, Hi Lindsay,
Thanks for your input. I really appreciate your help.
I did my size selection on a 2% agarose gel with a razor blade. As Lindsay said, it's cheap (and we don't have any other method available in our lab). When I put the results of my size selection on a gel I can see a light band around 400-500bp and nothing else. I certainly have RNA contamination but it shouldn't be amplified, right? And if the smirr is RNA contamination I should see it before the PCR?
We ordered some NEB Phusion for the PCR but right now I am testing my protocol with regular Taq. From what you are both saying, it could be possible that I have some shorter DNA fragments even after my size selection and those could be amplified preferentially by my regular Taq. I will try with NEB Phusion to see if it solves the problem.
You don't think it could be a problem with my ligation? You think it is related to my size selection and PCR?
it is really strange that all the six result showed a clear band with ~100 bp in size. do you think so? Maybe this indicates enzyme contamination in all the six RAD library? Is it possible that you have added the same enzyme in all the six samples, except for the specific restriction enzyme, which may digest the DNA more slowly?
Hi Ying Huang,
I have this 100 bp band in the negative control (not shown on the gel) even when I change my water and Taq Premix. I think this band is caused by primer-dimers.
To anyone who would be interested,
I realized that the protocol I was using suggested 1.5% Agarose gel to size-select the fragments. So that was what I was using previously and not 2% as I wrote before. I switched to 2% and now it's working fine!
Thanks again Lindsay.
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