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mutagenesis expert come in~


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#1 bry512

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Posted 30 September 2009 - 05:42 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?

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.

#2 Michaelro

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Posted 02 October 2009 - 02:25 PM

Hi
From my experience, sometimes, the PCR reaction doesn't wok efficiently and You don't observe the clear band of amplified product.
However, after transforming the competent bugs, You still obtain few colonies that carry the mutated plasmid.
If your competent bugs are of high quality, I'd take 2-5ul from PCR reaction for transformation.
If no colonies obtained or only wild-type colonies - then You should perform the troubleshooting over all the process.
Good Luck

#3 bry512

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Posted 04 October 2009 - 07:01 PM

Thank you for ur suggestion, i will have a try~

#4 Alinor19

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Posted 05 October 2009 - 03:03 AM

Hallo
In making point mutants I've always used the protocol suggested from Quick-Change from Stratagene.
NB: I DO NOT use the kit! I just follow their indications about primer design and PCR cycling.
Therefore I've never used 35 PCR cycles. I normally use 12-15 cycles.
And after this small number of cycles I never test on a gel (it is quite improbable that something can be seen).
But after the transformation in competent cells of 2-5 microl of DpnI treated solution I usually have the desired mutant.
Another big difference in ours protocols is that I use 125 ng of each primer /50 microliter mix. In your case, from a rough calculation,
there are about 2000-2500 ng.... Of course you are making many more PCR cycles, but I wonder if this could somehow affect your results....

One more note... I don't know which is your cloning experiment in detail, but usually when you clone in a MCS that is
inside the beta-Gal gene what you look for are the white colonies, not the blue ones! Are you sure that the white colonies
you obtained are not the one you are loking for?

I whish you the best for your mutants... :-)

PS: If you need the protocol of STRATAGENE it is on their site (or I can forward it to you if you prefere)

:)

Bye Bye

Edited by Alinor19, 05 October 2009 - 04:52 AM.





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