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| 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. |
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#1
09-30-2010, 03:01 PM
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 BamHI and SacI- double digest adviced against by NEB I'm digesting my DNA with BamHI and SacI for cloning. I checked NEB double digest finder for the buffer to use. Although NEBuffer 4 gives 100% activity for both enzymes, double digest is not recommended and sequential digestion is suggested. Why is that so? Has anyone done double digest against such advice by NEB? How did it go? Last edited by jlf; 09-30-2010 at 03:03 PM.  |
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#2
10-29-2010, 01:20 AM
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| Re: BamHI and SacI- double digest adviced against by NEB BamHI has star activity in Buffer 4 i.e. it may cut at sites other than GGATTC. I would do a sequential digest if I were you. Or if you are lazy and your lab is rich, buy the high fidelity version of BamHI that doesn't have star activity in Buffer 4. |
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#4
11-16-2010, 07:52 PM
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| Re: BamHI and SacI- double digest adviced against by NEB I will also suggest to buy high fidelity BamHI........ Rest double digest works fine....... |
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| adviced , bamhi , digest , double , neb , saci |
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