One of the undergrads in the lab has been trying to amplify a sequence of 450bp. In the resulting gel we get smearing all the way up to and over 6000bp. With an extension time of 60 seconds this shouldn't even be possible. This occurs in both in samples and positive controls.
One key issue is that the forward and reverse primers have annealing temperatures separated by 10C. We used a primer set from the literature. Bare minimum we should design our own primers, maybe even shorten our extension time.
However, I am captivated by the HOW of this situation. For my own curiosity, how is it possible to get over 6000bp amplification at 60 seconds.
First define the PCR conditions you used + the Tm of the primer set so readers would somehow have idea what is the problem.
However to give you initial advice, I think the extension time is too long for 450 bp, try reducing it to 10-15 secs. And another advice would be: do gradient PCR so you would know the optimum annealing temp; even though primer sets came from literatures. Conditions may be different from the expt in papers and in your lab. Anyways, readers are still waiting for the complete profile of your PCR condtions. Thanks.
If your got smearing over 6 kb, then it is not amplification, unless you can see a clear band at some high molecular weight. What you observed is probably smearing consecutive to performing a PCR with too much template DNA. I also agree with the previous message that you may optimize your PCR setup: salts concentration, Taq polymerase (brand and type), primers Tm, elongation and annealing time... If you could provide a picture of what you saw on gel, it would be better to further discuss. Thanks.
Hi,
how many cycles did he run? Too many will give You anything.
Here is the protocol for clarification. As stated, since we have wonky primers (uneven annealing temperatures), we have a wonky attempt to fix it. This is directly from the Undergrad's notes:
3 Min 95C
8 Cycles: 30 sec 95C, 30 sec 55C, 1 Min 72C
32 Cycles: 30 sec 95C, 30 sec 42C, 30 sec 72C
5 Min 72C
We definitely see smearing, but in Gaussian fashion. We had discussed the plausibility of too much template as well.
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