I'm trying to run a methylation-sensitive AFLP analysis and I only have hot-start taq (GoTaq).
I've seen in other AFLP procedures that you should not use hot-start Taq for pre-selective PCR because you need the Taq to ligate the adapters to form a double-strand.
So, will the pre-selective PCR not work if I use a hot-start Taq?
Thanks,
Stephanie
Dear Stephanie S Coster
It should work, you can try in small numbers of samples to avoid the wastage of Taq.
vikas
Dear Stephanie,
It may work, but first you have try in small quantity of your samples.
Good Luck
Hi Stephanie
It may rationally not work well because the 5' ends of the adapters you have ligated to the restriction derived fragments commonly have no phosphate and therefore only 3' ends are ligated. So when trying to pre amplify, the 3' ends of the fragments need to be filled-in by a common polymerase (Taq, Tth etc) at 72-75C for a few minutes. So it suggests having a PCR step at 75C for 2-5 minutes before the main steps of denaturation and other 25 (usually) cycles of pre-amplification. otherwise, if you have ordered special adapters having 5'-P then you don't need this step and can use hot start-Taq for preamplification.
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