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molecular cloning


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#1 vikrant saa

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Posted 10 May 2009 - 03:44 AM

I am planning to do cloning of a GST fusion protein in PGEX vector. I need to do restriction digestion of PCR amplified product with BamH1 and EcoR1 (restriction site present at 5'ends of primers ). I want to know how many additional bases BamH1 needed in addition to restriction site at the end of primer for efficient digestion or just restriction site is enough without additional bases.

#2 perneseblue

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Posted 10 May 2009 - 05:21 AM

You do need to add additional bp around the restriction site. Please check NEB technical guide on their website.
May your PCR products be long, your protocols short and your boss on holiday

#3 Vini

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Posted 10 May 2009 - 10:06 PM

I am planning to do cloning of a GST fusion protein in PGEX vector. I need to do restriction digestion of PCR amplified product with BamH1 and EcoR1 (restriction site present at 5'ends of primers ). I want to know how many additional bases BamH1 needed in addition to restriction site at the end of primer for efficient digestion or just restriction site is enough without additional bases.



Hi,
addition of extra bases is always advisable. as a general rule, I add extra 3 bases.




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