I have a big problem with my mutagenesis. I'm supposed to create 12 mutants, each of them one single point mutation. Unfortunateley it doesn't work and I'm very desperate now.
I designed my primers with the Quick change sdm programme. And all of them are above 80°C.And reverse and forward primers bind to the same sequence on opposite strands.
I have two plasmids one 8.5kb and the other one 7.9kb. So I make 6 mutations separatley in each of them. I followed the kit instructions (without having the kit, but having Pfu Turbo) and I could create 2 mutants in the 7.9kb plasmid and one in the 8.5kb very easily. After that eveything went down. I didn't get any colonies or I got some, which I couldn't recover in liquid media.
I begged my supervisor to buy the kit, but even with the kit it didn't work.
I started to optimise my PCR using normal Pfu and DMSO. And running the cycles 20 times (in the kit it says 12 times) to see if I can detect bands on agarose gel and yes I can (correct size). After DpnI (brand new enzyme) digest I transformed once into electrocompetent XL1blue and into chemical competent XL1blue cells. I got colonies and sequenced the area, but I didn't get any correct clones (they had a mutation, but on the wrong spot).
I thought I needed a more accurate polymerase and now I'm using the Phusion Hot start II high fidelity DNAP.
The protocol looks like that:
5x phusion buffer: 1x
10mM dNTPs: 200um each
primer A: 0,5uM
primer B: 0,5uM
Polymerase: 0,02 U/ul
4)72 4,25 min (30s/kb)
4 till 2 20x.
I digested with DpnI for 2 1/2 hrs and then purified with a PCR purification kit and measured with Nanodrop, so I still have 30ng/ul as concentration. I used 45ng for transformation. But I don't get any colonies.
I was thinking maybe I can increase the transformation efficiency if I ligate before I transform.
I have no idea what to do next.