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Problem in restrction digestion and ligation?

Problem in restrction digestion and ligation? - Molecular Cloning Forum

Problem in restrction digestion and ligation? - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 01-17-2010, 04:47 PM
Pipette Filler
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Default Problem in restrction digestion and ligation?



I need to clone one gene from Gram-positive bacteria into E. coli using pET-15b vector. Now, I have to digest them with Fast digest BamHI and XhoI enzymes (Fermentas). In Fermentas manual, it was mentioned that these Fast digest enzymes will cut the DNA in 15 min. Once, I subjected digestion for 4 hours at 37⁰C. For, pET 15b, I observed several bands. Can anyone help me, how much exact time I need to digest using Fast digest (Fermentas) BamHI and XhoI? Earlier, I cloned with normal enzymes at 37⁰C overnight and ligated at 16⁰C overnight. I had no problems then. It got cloned. Now using Fast digest enzymes, I had some proble of digestion time I fell. Please any one help me in this regard and also suggest me ligation mix? I have transform into DH5 alpha cells.
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Old 01-28-2010, 05:02 PM
Pipette Filler
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Default Re: Problem in restrction digestion and ligation?

I'm confused - if the Fermentas manual says to do the digest in 15 min, well by golly do the thing in 15 minutes. Why 4 hrs?

Furthermore, if it was easier to clone with standard BamHI/XhoI then use standard BamHI/XhoI. Do you have no choice which enzymes to use? Surely some lab in your department has the regular enzymes and would be willing to let you borrow a little.
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