mutagenesis expert come in~ - (Sep/29/2009 )

I want to do a site directed mutagenesis in a 6kb plasmid, the primer set is
primerF: GCCTCTGCTTAGTGGaaTTcCCACCCCCACAACCCGCAGG
primerR: CCTGCGGGTTGTGGGGGTGGgAAttCCACTAAGCAGAGGC,
The reaction system was designed as following:

template 1ul (100ng)
primer 1+1 ul (10mM each)
dNTPs 1 ul (25mM each)
10Xpfu buffer 5 ul
pfu 1 ul
H2O 39ul
total 50ul

95C 2min
95C 30s 35cycles
68C 50S
72C 6min
72C 10min
After 1ul DpnI digestion, I load all the 50ul PCR on the gel, however,I could not see any obvious band.
Could anyone tell me what is wrong?

The first thing I would try is reducing the annealing temperature from 68 to 60 or to 55 and retrying the PCR. I believe the protocol is to cycle for only 15-18 cycles and directly transform, which I would suggest trying with a lower annealing temperature. How were the primers designed? How long is your plasmid?

I would also try adding 5% betaine to the reaction, especially if the rest of the plasmid DNA is of similar high GC content.

Did you try to transform? I never check my point mutation by gel, as I understand you dont have to see a band even if your mutation was ok

bry512 on Sep 29 2009, 10:40 PM said:

I used the softwere to analyze the primers, no secondary structure were indicated. The calculated Tm is around 85-92C, and the forward and reverse primers are exactly complementary to each other. That is why I tried 68C as the annealing temperature.

I tried to transform up to 10ul of Dpn1 cut PCR, but I havenot got blue colonies with x-gal/IPTG colour screening (white colonies i got, but not many) However, I have not tried transform all the PCR. Maybe I need to try.

I heard a clear band after DpnI cut should be observed, but in my case I havenot observe obvious band, that is why I suspected that the mutagenesis PCR is not working.

Why do you think blue colonies are necessary (or even possible?). Have you checked the white colonies you have for being the correct construct?

i just follow the instruction of the stategene kit. But I checked again the instruction, the colour screening should be only for the control plasmid. I think i wrongly used that for my own mutation.
I think I need to first try transform all the PCR to see whether I can get any mutation, and if it still not work, then optimize the PCR. Do u agree?

I would pick some of your existing white colonies, grow them up, miniprep and seequence them. You probably already have the results you want.

I did the mutagenesis once again, this time I changed the annealing temperature from 68C to 60C.
After PCR I use Dpn 1 to cut for 1h, and then use ethanol and sodium acetate to precipitate all the the DNA, the precipitated DNA was dissolved in 10ul water.
I transformed all the 10ul DNA, finally I got around 100 colonies. I used the vector primer to sequence, and the template plasmid for the mutagenisis as the positive control of the sequencing. I have sequenced 12 colonies, but none of them gave out signal in the sequencing reaction. wheras the positive control has the good signal.
So do u suggest i should pick more colonies for sequencing, or troubleshooting the mutagenesis PCR?

I don't understand what you mean by "gave out signal." Do you mean the sequencing failed, or that the sequence is wrong (same as the parent vector)?