Problems with Site-Directed Mutagenesis - (Apr/29/2011 )

So I'm doing site-directed mutagenesis to change a GC pair to an AT pair. The problem is that I don't see any colonies growing on LB-agar plates after transformation. My plasmid + vector = ~ 10 kb

I did PCR for 30 cycles, 25 uL total volume (I've tried 19 cycles before, still didn't work)

4 uL 100 ng/uL tempate
1 uL each of 50 uM forward and reverse primer (39 bp each)
2 uL 10 mM dNTPs
3.75 uL 50% DMSO
5 uL 10x pfu bf
1 uL Turbo pfu hotstart (Stratagene)
fill volume with RNase, DNase free water

Denature, 95 deg C - 5 min
Denature, 95 deg C - 1 min
Anneal, 55 deg C - 1 min
Extend, 72 deg C 0 20 min
Final Extension, 72 deg C - 20 min
4 deg C forever

After PCR I digest it with 1 uL DpnI, 1 hr, 37 deg (I've tried 1.5 hr, and I still got no colonies)
Transform the digested product into 100 uL competent E. Coli cells (5 uL of DNA, incubate on ice 30 min, heat shock 42 deg C for 30 sec, ice 2 min, 300 uL SOC medium, shake at 225 rpm for 1 hour), and inoculated on LB-agar plates. Let bacteria grow overnight at 37 deg C.

When I check the plates the next day, I get no colonies. I've done this procedue with a positive control that DID work - there were like 5 colonies and the conc was about 160 ng/uL on the Nanodrop. I tried gradient PCR (anneal 45 - 55 deg C) but still got nothing. One time my sample plate had a single colony, but it turned out to be WT DNA, and not my mutated one. I checked my template on a gel, and looks like there is still intact DNA there at the right size.

I'm actually starting to think that the problem may be my primers? Because my positive control worked before. These are my primers:


WT Forward Sequence
ctg gag ctg ctg gtg gcc gtg ggc ccc gat gtc ttc cag


Designed Mutated Forward Primer

ctg gag ctg ctg gtg gcc Atg ggc ccc gat gtc ttc cag


WT Reverse Sequence
Ctg gaa gac atc ggg gcc cac ggc cac cag cag ctc cag


Designed Mutated Reverse Primer

Ctg gaa gac atc ggg gcc cat ggc cac cag cag ctc cag

Are they too GC rich? Do you have any suggestions for redesigning primers?

Someone in the lab has done mutagenesis a LOT, so the above is her protocol that I followed. The template is the same template she's been using. She has never been unsuccessful.

What am I doing wrong? Help is appreciated!

Hmm...

I'm also in the middle of site directed mutagenesis. The only differences between our protocols is that you use 400 ng of template while I use 5ng. 400ng seems like a lot of template, to me. What concentration are your primers at? I would suggest you decrease the amount of template you use and increase the primers. Additionally, why not try your PCR in a 50uL volume?

i usually use 50 ng of template and 125ng of each primer..., it worked fine for me....i would suggest you to check the competent cells, are they good enough??? 5 colonies in 160ng of plasmid doesnt seem to be enough competent upto me... then after Dpn digestion i do gel extraction and then transformation.

good luck,
Gnana...,