Site-directed mutagenesis problem
Pepa Florez Pérez
(by snipeurope from hotmail.com)
Tue Jan 18 07:16:12 EST 2011
I am writing to you becasue I am experiencing problems to carry out a site-directed mutagenesis.
I use two oligonucleotides 22 nts. long, that hybridize completely but not on a single-base located in the middle of the sequence.
What I get after carrying out the PCR is a smear in the gel:
- The enzyme is not a problem since the positive control (another plasmid same lenght with different oligos did work)
- I get a smear no matter which vector I use as template, if I use the new oligos, but not the others.
- If I increase template concentration I get the same result.
Can you guess what it is going on? What else would you try? I am really lost.
If any of you is interested I can send you a summary picture too.
Thank you very much
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