I'm trying to do a long-range PCR (~10kb) and I'm just wondering what polymerase to use despite the Q5. Do you have any experience in doing a long-range PCR with other polymerases? Did you use the standard protocols listed in the manuals or did you adjust the protocols (if you did it, what did you exactly adjust? Time or temperature?)? And last question: Did you use a MasterMix?
Thanks for your help!
Hi Iris, we use SequalPrep™ Long PCR Kit with dNTPs (Thermofisher) for 8-10 kb long range PCRs and it work fine (its a kit, Mastermix and Taq). We have tried both enhancers and the enhancer B worked a little better, we also adjusted the DMSO % (but this modification was due to our primers) and I think that was all. We used the manufacturer's recommended program.
Here you have the link:
https://www.thermofisher.com/order/catalog/product/A10498
Good luck!
Hello, Iris!
In our lab we use BioMaster LR HS-PCR kit by Biolabmix.
Here is the link: https://biolabmix.ru/en/catalog/nabory-i-smesi-prc/prc-long-fragments/biomaster-lr-hs-pcr-2x/
It works out-of-the-box without any adjustments of provided protocol. We get good results in obtaining amplimers up to 17 kb (we don`t need longer pieces but manufacturer says it is possible to get product up to 30 kb).
I also added electrophoregram of one of our latest results (top band of the DNA ladder is 10 kb).
Before that we used Expand Long-Range PCR System by Roche but it was a little "capricious" and sometimes failed to generate good amplimers despite same conditions of PCR.
I hope this information will be helpful!
Good luck!
I used VENT polymerase from New England Biolabs according to kit instructions with minor modifications. In brief, set up 2 25-ul reactions instead of 50 ul. The lower volume reactions worked better in our thermocycler than the larger volume reactions, likely due to heating efficiency.
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