I know similar questions have been asked before but a lot of the answers simply state what the manufacturers say about the polymerase or haven't been able to amplify over ~14Kb. So I have three fragments I want to amplify, 14, 16 and 17kb (human genomic DNA), has anyone successfully amplified fragments this long? If so which polymerase did you use? Has anyone successfully amplified this length using Phusion, NEB's LongAmp hot start or Sigma's AccuTaq LA?
I would like to be able to use the TAKARA LA polymerase but am struggling to get TAKARA to respond to my emails and they dont have a UK phone number.
(Any other general advice for this length amplicon would also be appreciated)
I haven't done a lot of large fragment amplification but I have heard others say this Phusion Hot Start Polymerase works well. Here is a link:
https://www.thermofisher.com/order/catalog/product/F549L
Good Luck!
Hi,
For medium range (8-15kb) you could try the GoTaq Long PCR MasterMix offered by Promega. For larger fragments at least up to 20kb the LongAmpTaq provided by NEB shows a high fidelity. I used both polymerases for the amplification of fragments from plant gDNA. The critical point in such experiments is the isolation of high molecular weight DNA. Since this might be easier from human samples, your chances of success are high.
Good luck!
Dear Hazel,
i would recommend PrimeStar GXL for high fidelity PCR of upto 30 kb region. You can reach us at [email protected] for exact query and one of our UK representative will contact you afterwards. Please don't hesitate to contact us.
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