You could do a sequential digest. Do digest one, purify and do the digest with the second enzyme.

You can try step by step digest

You could do a sequential digest. Do digest one, purify and do the digest with the second enzyme.

what Restriction endonuclease are you select？

Dear khan,

What kind of enzymes you are using and could you please provide the information about buffers you are using in digestion process.Basically buffers place a major role in digestion.please check the concentration and receipe of buffers,use lower concentration first .

perform the digestion with one enzyme first, run on agarose, excise the band, purify the band from the gel, estimate and then perform the second digestion.

I suggest using NEB's double digest finder to identify which buffer to use. Even if you're not using NEB enzymes, it will help you to identify which enzyme's buffer you should use. Of course, not all double digests can be done simultaneously, but NEB's site will identify those too.

http://www.neb.com/nebecomm/DoubleDigestCalculator.asp#.UO2ba3d4wxA

I agree with these suggestions. If performing sequential digest is too much for you, then I recommend checking the buffer compatibility information sheet from your restriction enzyme provider, for the buffer in which both enzymes will retain most activity. You could extend digestion time and use a higher enzyme:template ratio to improve chances of complete digestion.

Try fast digest enzymes (Thermo) if enough money for ordering is available! Worked well in my hands.

If sequential digest (as already suggested) is a problem try to amplify the insert of interest with primers containing sequence overhangs for restriction enzyme cleavage sites you need to get in frame ligation. Add 3-4 additional GC's to get sufficient digestion of PCR product.

Good luck,

Kai

you select the nearest matching restriction buffer in which one of the enzyme shows 100% activity and another enzyme at least shows 50 % activity in selected buffer. Add the double amount of enzyme showing 50% activity.

Alternatively you can got for the choice of fermentas fast digest enzyme where you will find single buffer compatible with all enzymes

Thanks to all for the Kind replies! These are really informative. Actually I had compatible buffer but one enzyme had only 10-25% activity in this buffer while other has 100%. As Sachin replied, i have tried this too.

Would you mind revealing what result you got after doubling the enzyme having lower activity in the buffer used for double digestion?@ Muhammad Faheem Khan