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mutagenesis expert come in~


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11 replies to this topic

#1 bry512

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Posted 29 September 2009 - 07:40 PM

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?

#2 phage434

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Posted 30 September 2009 - 04:11 AM

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.

#3 laurequillo

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Posted 30 September 2009 - 04:17 AM

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
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#4 fishdoc

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Posted 30 September 2009 - 05:33 AM

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?

#5 bry512

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Posted 30 September 2009 - 05:35 PM

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.

#6 phage434

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Posted 30 September 2009 - 06:45 PM

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

#7 bry512

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Posted 01 October 2009 - 01:23 AM

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?

#8 phage434

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Posted 01 October 2009 - 05:28 AM

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

#9 bry512

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Posted 08 October 2009 - 12:38 AM

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?

#10 phage434

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Posted 08 October 2009 - 04:00 AM

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)?

#11 bry512

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Posted 08 October 2009 - 07:06 PM

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

#12 phage434

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Posted 08 October 2009 - 07:34 PM

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.




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