I need to optimize a long range PCR that uses consensus primers to amplify a diverse intervening segment. The segment to be amplified differes for the different bacterial strains and ranges from 11kbp to 20 kbp. I am using iProof with the buffer without GC enhancer (my target is low in GC).
First I used the recommended master mix consitituents as in the protocol and an extension time of 15sec/kbp (kit recommends 15/30sec/kbp), taking 20kbp as the length of expected product as this is the longest I expect.
I got a 11kbp product for the shortest of the expected products but the other two sizes did not give any bands. Then I extended the extension time to 30sec/kbp and I got a product for the intermediate strain (15kbp) but not for either the shortes (11 kbp) or the longest (20 kbp).
Now I have doubts if I can optimize a uniform thermocycling profile that may be able to amplify all expected products or not! My next option is to try gradual increase in the extension time as suggested in the Qiagen extra long PCR kit protocol and see.
Any suggesions/ comments are welcome.
Long range PCR optimization with consensus primers and different product lengthsLong range PCR consensus primers
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