11 replies to this topic

[bry512]

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?

[phage434]

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.

[laurequillo]

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

[fishdoc]

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

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?

I've never done site directed mutagenesis on a plasmid, but is it detrimental that you two primers are exact complements of each other? Would that level of primer dimerization severely inhibit a PCR reaction?

[bry512]

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.

[phage434]

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

[bry512]

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?

[phage434]

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

[bry512]

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?

[phage434]

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)?

[bry512]

I mean failed to sequence for the selected clones, while positive control worked.

[phage434]

Well, I'd say you have no information.  Your minipreps of the transformants may have failed, you may have incorrect primers, or you may have a different problem with your sequencing.