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Problem with restriction digestion - (Sep/29/2005 )

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[quote name='berwal' date='Sep 30 2005, 01:27 AM' post='26282']

OK that's make sense can you send the primers as a text file ?
You didn't answer the question Did you sequence the clones ?

You should always check your clones cause even if the Taq error rate is low there's still some chances to get some messed up clones believe me I'm doing this type of stuff for 20 years wink.gif

Let's start to look at the primers then will see the other hypothesis

No Sir I have not yet sequenced the clone as it takes a lot of time(2-3 weeks) here at an institute closeby. We don't have a facility with us.

OK I'm not in the lab right now but I can see the XhoI site and it's not that proximal to the end so it should be easily cut out even from the pCR product well anyway.

I understand that of course if you don't have a sequencing facility clause to you it's difficult. Here is what I proposed to you :

Start a new culture and use right away the Quiagen kit and please don't forget to use the optional PB buffer (which remove endonulease) and run several digest

BamHI and XhoI (they sould work in buffer NEB2)
BamHI and NdeI
NdeI and XhoI
Also use a control DNA to exclude thst they're any problems with each single enzymes



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