Im having trouble with site directed mutagenesis, and after attempts at trouble shooting with no luck, hoping someone might have some advice. The PCR mix includes:

- 1uL of plasmid DNA[50ng/uL] at 5kb in size (~48% GC)

- 5uL of dNTP mix[8 uM]

- 2.5 uL of forward and reverse primers [10uM each] that are ~40bp and Tm ~62oC

- Im using Q5 polymerase (1uL) with Q5 buffer (10uL), and ddH20 for total of 50uL.

- negative control includes all this but no primers

I will then take 20uL of my PCR reaction and incubate with 1uL of DpnI for 5-6 hours at 37oC. Once finished, I transform 100uL of Chemi Comp cells with that 20uL solution, recover for typically 1 hour (I've tried longer), and plate with my appropriate antibiotic.

I've tried increasing DNA-template concentrations, longer extension times (up to 10 mins), varying annealing temperatures (56-68oC), increasing the amount of cycles (typically 20 cycles, but also tried 30), and I still have no luck. I either get no colonies on my plate (but my negative control does), or when I do and send out multiple colonies for sequencing, the mutation is either not incorporated, or there is non-synonymous mutations elsewhere.

I appreciate any help/advice I can get!