Hi All,I am trying to amplify a 5Kb fragment from genomic DNA.
I have designed 26-28 bp long primers(tm ranges from 61-64 C) and have tried My Taq mix from Bioneer and long range PCR kit from QIAGEN, still unsuccesful.
Could somebody guide me in optimizing my PCR?
The PCR reaction mix for My taq kit from bioneer is presupplied and you just need to add template and primers, however for long range PCR kit, my reaction mix constituted of 10X buffer,500um dntp's, enzyme, 5X Q solution(enhancer), template and primers.
if the other things are as per set protocol, most probably the problem lies with the genomic DNA. it should be intact especially in the region to be amplified. if not already done, you can try re-isolating genomic DNA.
Hi Satparkash,
I can vouch on the DNA's purity as the nano drops A260/280 ratio ids 1.8 and it gives an intact band on agarose gel.
Whats is the PCR condition? This PCR product length needs nearly 8 to 10 minutes for PCR extension time.
Well I am giving an extension time of 5 minutes as per 60sec\kb.
Do u think taq polymerase requires greater than that?
I think 5 minute is not ok. try to increase both denaturation to 3 min and extension time to 8min. after getting positive results try to decrease time for optimization.
You may also need to change annealing temperature for better results.
In this case you also need to use special taq that has activity for long range PCR. at first step see the manufacturer's protocol for taq polymerase activity if it covers 5 kb in length.
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