Though my PCR is successful with DNA Phusion polymerase, I am still unable to get transformed colonies post KLD assay with my DH5alpha comp cells. What can be the possible reason for this? I am using pET28a vector, doing gel extraction prior KLD, and following NEB protocol.
OK, lots of possible ways this could have gone wrong. Let's see if you can narrow them down.
Have you had success with transformation in the past?
Did you check the vector on an agarose gel after PCR?
What volume of vector did you add to your cells?
What controls did you use - transform cells with pUC? Smear "transformed" cells on non-selective media?
Are you doing electro- or chemical- transformation? If electro, did you get an arc or spark (=dead cells)? If chemical, did you make 100% sure the heat blocks are at the correct temperature?
Anyone else in the lab using these cells? If yes, does it work?
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