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Problem of time required for complete digestion using Fast digest enzymes

Problem of time required for complete digestion using Fast digest enzymes - Molecular Cloning Forum

Problem of time required for complete digestion using Fast digest enzymes - 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, 05:07 PM
Pipette Filler
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Default Problem of time required for complete digestion using Fast digest enzymes



I need to clone one gene (1.6 kb) from Gram-positive bacteria into E. coli using pET-15b vector (>5 kb). Now, I have to digest them with Fast digest BamHI and XhoI enzymes (Fermentas). 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 used normal enzymes to clone some other genes. I had no problem. But now, I got problem with time using Fast digest enzymes? when I tried the same gene with normal enzymes (not fast digest), single bands were seen. After ligation, there are several colonies and had no insert in them. Now, I had no normal enzymes with me. I had only Fast digest enzymes. Suggest me time for Fast digest enzymes?

Last edited by srivels; 01-17-2010 at 05:11 PM.
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Old 01-25-2010, 07:49 PM
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Default Re: Problem of time required for complete digestion using Fast digest enzymes

Fast Digest enzymes digest FAST! How much DNA are u taking? Read the product insert- typically 1ul of enzyme will digest 1ug DNA (in 20ul) in 5 mins. If you have too much enzyme and too less DNA and digest for 4 hrs, there will be much non-specific chewing up and there's your problem!
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