|Register||Search||Today's Posts||Mark Forums Read|
|Molecular Cloning Forum 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.|
| ||LinkBack||Thread Tools||Display Modes|
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.
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.
|digestion , ligation , problem , restrction|
|Thread||Thread Starter||Forum||Replies||Last Post|
|DNA Ligation Problem Double Digestion||Denise||Molecular Cloning Forum||8||02-21-2012 05:40 AM|
|Problem of time required for complete digestion using Fast digest enzymes||srivels||Molecular Cloning Forum||1||01-25-2010 06:49 PM|
|Ligation||Deitiker, Philip R||Protocols and Methods Forum||1||06-28-2007 12:40 PM|
|Ligation||Deitiker, Philip R||Protocols and Methods Forum||0||06-21-2007 11:11 PM|
|Ligation||Deitiker, Philip R||Protocols and Methods Forum||0||06-21-2007 09:12 PM|