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Stratagenes Dpn1 cleaves my PCR product


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#1 linli

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Posted 20 August 2010 - 04:11 AM

Hi!
I have been having problems with Dpn1 digestion. I have preformed site directed mutagenesis and after that I want to break down my start template with Dpn1. The problem is that I do not get complete digestion with Dpn1. Of four samples sent on sequencing so is approximately 25-50% wt, which means that Dpn1 digestion has failed. So than I thought that it must be something wrong with my Dpn1 (from Fermentas) so I tested Dpn1 from Stratagene. Then I got even stranger results, se gel results in the attached file. It seems like digestion with Dpn1 from fermentas give a visible PCR sample on the gel while the same PCR sample digested with Dpn1 from stratagene is not visible on the gel. Confusing results for me, does anyone know if it should be a difference between Dpn1 from Stratagene and Dpn1 from Fermentas? PCR product that was not ran in the temperature cycler (No PCR, negative control) is not visible on the gel so it must be amplified PCR product I see on the gel. But then it seems that Dpn1 from Stratagene breaks down PCR product which should be impossible or?

I add 1ul Dpn1 directly to 50ul PCR (site-directed mutagenesis) product and incubate in heating block at 37C in 60min.

Does anyone know how good cleavage I can expect with Dpn1, how many percent in a sample is wt? I work with saturation mutations and screen libraries so I need to have as less wild type as possible.

Does anyone know any better strategy than Dpn1 digestion to get ride of the start template after site directed mutagenesis?

Im very thankful for all the help I can get.

/Lina

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#2 NemomeN007

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Posted 20 August 2010 - 07:14 AM

Well, you're still getting your mutation, so I wouldn't call the procedure a failure (1 or 2 out of 4 is not bad), so the technique is working. Also, DpnI is not the most efficient enzyme either (especially with hemimethylated DNA), so I wouldn't assume that it "failed" because you are getting 50% success. Rather, I would go on the premise that perhaps using Quickchange to do your studies may not be the best approach.




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