PCR large fragments - (Apr/26/2006 )
has anybody amplified large fragments >7,5 kb ? I need to amplify a large fragment, but error rate has to be very low, so I don't get any mistakes into the amplified sequence?
Try pfu ultra or phusion enzymes. There are kits for long extension pcr, and it is probably worthwhile to read the manuals for those, but probably not worthwhile to buy the kits. Check Invitrogen and Stratagene. Don't use Taq or Taq mixtures ("High fidelity" enzymes).
I use for 13kb the Eppendorf triple master. I've not sequenced all the fragment. But in 2500pb i don't have any error... (12 different sequencing) So i can't tell more for that kit.
I've succesfully the expand plus pcr kit for 15kb amplifwith reat success. But as says phage 434, it may not be necessary to buy that kit.