I am trying to amplify a 1.8kb insert from a clone, using Pfu. I have standardized the conditions by using Taq. i used a graditent from 50-60 degrees, and i got the desired amplicon for all temperatures. the weird part is reaction just does not seem to work with pfu. i have tried changing template concentration from 1 pg-500g, i have varied MgCl2 concentration as well, and i have also tried using a gradient. i am completely and totally out of clue as to what might be going on. i have also tried Vent and Deep Vent, without any luck. Please Help!