Edited by donny, 30 September 2010 - 07:02 AM.
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5 replies to this topic
#1
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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?
#2
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Growing old is mandatory, growing up is optional...
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).
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.
#3
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Posted 30 September 2010 - 12:47 PM
The costs are identical, not higher.
#4
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Post Dog
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.
Cheers,
rsm
Cheers,
rsm
I got soul, but I'm not a soldier
#6
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Unlimited ligation works!
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
May your PCR products be long, your protocols short and your boss on holiday
