PCR product of mutagenesis_good but still wrong

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roxanalibe
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PCR product of mutagenesis_good but still wrong

Post by roxanalibe » Tue Jul 20, 2010 1:44 pm

Hallo,


I have this problem introducing a point mutation in an insert, with primers recommended by PrimerX_ Primer Design Based on DNA Sequence. I know the primers are good, as I introduced the same mutation in the same insert but different vector. I cannot cut it from there and put it in the vector I need, as the insert in which I want the mutation has also GFP fused to it, so it is more special.

The thing is that in the last month I tried several approaches (I use QuickChange Site Mutagenesis Stratagene kit, with Pfu Turbo enzyme and I followed their protocol, playing with the amount of DNA and primers). In fact last week I got my mutant (1 positive clone), perfect digestion size (compared with wild-type) and also the mutation was present (by sequencing) BUT in the sequence there is something wrong: there is a duplication of the fragment of the primers that contain the mutation, it is like the first fwd primer is followed by a second one, linearly. Of course they dont overlap perfectly so, they are not in frame; and the primers did not bind anywhere else, as I sequenced the full length of the insert... i never got this weird stuff, although I obtained couple of different mutants until now. I must say that I used for this last mutation (the only one that was positive) 2.5X the amount of primers that Stratagene recommends (I read on some forums that if you have problems with mutagenesis, getting only the WT form, you should increase the amount of the primers and dNTPs)

Now I need to start again with the mutagenesis, but if anyone has an explanation why I can get very easy the same mutation in a different vector, and not in the one I want, please let me know.

Thanks a lot for the input,
RA

roxanalibe
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Post by roxanalibe » Tue Jul 20, 2010 5:15 pm

ok, my boss unlighted me, and hope is this the answer to my problem: the miniprep quality could be affected in the purification process and by seq the DNA it can appear a duplication but on different frames (I am always using 3 frames way to read the DNA sequencing), and what can appear to be a duplication it can be in fact an artifact due to the low DNA quality, although there seems to be the mutation and the digested product is good....so, I need to transform new cells with the clone that I got positive and to make a good miniprep, and eventually maxiprep. hopefully, this will be a happy end story.

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JackBean
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Post by JackBean » Tue Jul 20, 2010 5:49 pm

that doesn't seem much rigth to me. How does look your sequencing look like? How many bp from the mutation are your seq primers?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

roxanalibe
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Post by roxanalibe » Mon Jul 26, 2010 9:13 am

it might be something wrong, as there is no way I can get a normal pattern for none of the miniprep I did from the transformation plate with the initial positive clone. the primers for seq are 100 bp distance from the mutation....still, I think I need to repeat the mutagenesis...and I begin to get sick of it :-(

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JackBean
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Post by JackBean » Mon Jul 26, 2010 12:58 pm

I understand that. Last fall I was doing all that stuff all the time, because I couldn't even clone my genes :( There was always plenty of mess...
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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