I am performing Kunkel mutagenesis using three (non-overlapping) primers to introduce soft randomizations into my protein of interest. Based on an agarose gel it appears the reaction has occurred (not entirely sure if correctly or efficiently) but following electroporation and DNA purification sequencing results reveal only template. Template plasmid contains TAA codons at all the positions to be randomized.

I am not sure how to improve the efficiency to ensure correct Kunkel mutagenesis of the template to produce my phage library.

I have attached a gel displaying the results of my attempt to optimize the experimental conditions:

1) varying annealing temps during initial primer annealing

2) increased [primer]

3) increased incubation at room temperature (following enzyme addition)

4) all three previous conditions

5) 2log DNA ladder

6) increased [dNTP]

7) dU-ssDNA template

8) previous unoptimized conditions