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BamHI and SacI double digest


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5 replies to this topic

#1 donny

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Posted 30 September 2010 - 06:54 AM

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?

Edited by donny, 30 September 2010 - 07:02 AM.


#2 hobglobin

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Posted 30 September 2010 - 07:11 AM

I guess it's because of star activity of one of the enzymes with NEBuffer 4. NEB offers high fidelity enzymes of both REs, you may want to use (hough the costs are higher).

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#3 phage434

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Posted 30 September 2010 - 12:47 PM

The costs are identical, not higher.

#4 Rsm

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Posted 30 September 2010 - 11:14 PM

Yes, I did a double digest of BamH1 and Nhe1 (in Buffer 2). Not recommended, but I had no problems. I also think that star activity might be the reason for this. I'd recommend to use max 5% of your total volume of RE (ie 0.5ul of each BamH1 and Sac1 in 20ul total volume) and increase restriction time to 2hrs at 37C. Should be working.

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#5 Prawn

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Posted 01 November 2010 - 07:45 PM

.......

#6 perneseblue

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Posted 02 November 2010 - 06:41 PM

Yup.I have conducted double digest with BamHI in buffers 2, 3,and 4 with no ill effect. I think as long as volume of BamHI is below 5% of the final digest volume, star activity can be avoided with BamHI
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