Previously I successfully amplified 1.7kb and 1kb using same method.
My Primers are 30nt in length and Tm=79 and 80. I used 200ng of gDNA.
My PCR condition is 94 for 5m, 94 for 30sec.
Annealing and extension I combined and tried at 68C and 72C for 1m:30sec, final extension for 10m and 30 cycles.
I am getting multiple bands, I attached the result file
Can I get some ideas please.
Hi Rukhsana,
To me it seems that your stock of gDNA has degraded. You should go for a fresh isolation of gDNA and set up a new PCR with it.
Best of luck this time.
few options:
1. as Anirban suggested, make a fresh stock of DNA.
2. lower the annealing time to 1min
3. change the enzyme. use a high-fidelity enzyme (phusion Taq works best for me)
4. check your primer sequence using the primer-blast server. note that even sequences with 3-4 mismatch bases would possibly be amplified in the reaction,
I don't know what your project's aims, but i would suggest ignoring the non-specific band and extracting the correct size from the gel using a gel purification kit.
good luck!
Promoters tend to be high %GC, which may also mess up PCRs, sometimes quite erratically. I've had my fun with a sequence that was about 80% GC, and bands would sometimes appear, other times not, using the same batch of reagents etc. Strangely, adding less substrate improved the reliablity of the reaction. If anybody has an explanation for that, I'd like to know (these were amounts
It's not really strange, Berlinda. When preforming PCR you rely on that the first cycles create small amount of the correct amplicon from the template, and that later cycles will amplify that product. in fact, when PCR works as it should, the polymerase and primers will work mostly on the product of those first few cycles, generating a single correct band. by lowering the amount of template DNA you make sure that the amplification stage of the process will work on a hopefully correct amplicon.
When using large amounts of template, the reaction products will derive mostly from the inserted template and in the case of more than one possible product, the polymerase activity will be "shared" between the wanted and unwanted products. this will show in the gel analysis as a non specific product.
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