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problem with site directed mutagenesis


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#1 biolab12

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Posted 13 December 2009 - 02:41 AM

Dear all,

I am doing a straightforward site directed mutagenesis to change one amino acid to another. But somehow I get no colonies after transformation. My PCR setup and conditions are as follows

1. Template DNA ( about 100 ng) 1l
2.10 X Pfu buffer with MgSO4 5l
3.dNTP mix (0.2 mM final) 1l
4. forward primer(0.2 M final) 1l
5.reverse primer(0.2 M reverse) 1l
6.Pfu polymerase (2.5U/l) 1l
7. Sterile water 40l

PCR conditions:

95 C for 3 min--------initial denaturation(1 cycle)

95C for 30 sec
54C for 1 min
72C for 8 min

repeated for 18 cycles

72C for 15 min--------final extension(1 cycle)

and final hold at 4C

Additional information:

1.My construct is 8.7 kb
2.The Tm of both the primers on the delivery report was 59 degrees. So I used 54 degrees as the annealing temperature.
3.I made 18 cycles hoping that it would be sufficient for the amplification with 72 degrees extension (Pfu polymerase purchased from fermentas) and 8 minutes extension time. approx(1min /kb)
4. Template DNA was removed with 1.5 l DpnI (2 hrs incubation at 37 degrees )
5. I use chemically competent cells for transformation. I checked for the efficiency of transformation with standard plasmid and seems to be ok.

I am not too sure if the problem is with the PCR reaction mix or PCR thermocycling conditions or with the transformation. How can I set up control reactions to make sure which step is going wrong? Any suggestions on the extension time used?

I would highly appreciate your expert advice.


Regards,
biolab

#2 phage434

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Posted 13 December 2009 - 05:58 AM

I would suggest making sure your pcr was working. I'd do this by first diluting the template 10x and increasing the number of cycles to 24 or so, then comparing the gels for the pre and post cycling of the reaction. Assuming your pcr is working, then I'd bet the problem lies in the transformation efficiency. Test this by transforming 10 pg of your favorite plasmid and counting colonies. You should get 100-1000 if your transformation is acceptable (plate small amounts or dilutions, you don't have to count 1000).

#3 biolab12

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Posted 13 December 2009 - 10:15 AM

thank you for your suggestion. I have some questions..

1.Does increasing the number of cycles to 25 or more affect the mutagenesis in anyway? (extra mutations or so)

2.Also in my case should I try increasing the extension time (Pfu polymerase from fermentas recommends 2min/kb length of the plasmid.)

3.In that case if I have extension time of about 16 minutes per cycle for 25 cycles , would the Pfu polymerase still be effective?

4.Also I ran a gel after my PCR (before DpnI digestion) and could see a very faint band near 8 kb but a comparatively bigger band at a much lower length (3 kb). I guess the faint band was the template I used, not too sure about the other band...


Would be great if you can clear these doubts!

#4 phage434

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Posted 13 December 2009 - 10:54 AM

Yes, I would definitely try an extended extension time. Your 3 kb band is troublesome, and may indicate priming at a second site other than the one your primers are designed for. Why are you extending only 8 minutes when the suggestion is 16 minutes?

Extra mutations will only occur if the amount of amlification increases. By cutting down on template and increasing cycles, you are doing some of that, but there is still way more template than in a typical pcr reaction, and the dNTPs will likely be limiting the amount of product made.

Pfu and other pcr enzymes are remarkably robust for long periods at high temperatures. The dNTPs (particularly dCTP) are the problem.

#5 biolab12

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Posted 14 December 2009 - 11:41 PM

I tried the mutagenesis again and did not see any band on the gel. I have done some successful mutations in the past but I never checked an aliquot on gel after the reaction. Do you normally get a bright band ?

I am just wondering what could possibly go wrong!

The primers are designed according to the Quikchange protocol...They are 31 bases long, both complementary and carrying the desired mutation in the middle, ends in one or more G/C. I guess my primers are ok..What is your opinion? (mutation shown in bold)

5-CGCTTATGGCCAACTATTCCTGCGTTTTCCC-3 (forward)
5-GGGAAAACGCAGGAATAGTTGGCCATAAGCG-3 (reverse)

I sequenced the whole plasmid . So the primers must, in theory, bind to my template...

I am going to transform today anyway and perhaps make new reactions changing the template amount.

Hope it works...




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