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BamHI and SacI- double digest adviced against by NEB

BamHI and SacI- double digest adviced against by NEB - Molecular Cloning Forum

BamHI and SacI- double digest adviced against by NEB - 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 09-30-2010, 03:01 PM
jlf jlf is offline
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Default 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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Old 10-29-2010, 01:20 AM
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Default 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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Old 10-29-2010, 12:31 PM
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Default Re: BamHI and SacI- double digest adviced against by NEB

Do the double digest. It works fine.
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Old 11-16-2010, 08:52 PM
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Default 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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