How to choose HindIII/EcoRI double digest buffer? - a stupid question, but I really curious about the answer (Apr/08/2006 )

Both New England Biolab website and catalog recomend to do the HindIII/EcoRI double digest in E.coRI buffer. But the HindIII only have 50% activity in that buffer. Actually, it is gonna be better if I do the double digest in NEbuffer2, where both enzyme have 100% activity?

EcoRI displays star activity (cuts at AATT sites) in buffer 2, suppressed in EcoRI buffer. We do it anyway.

You should be careful about ligation and transformation of DNA cut in EcoRI buffer -- the triton X-100 trashes the transformation efficiency of chemically competent cells, even in very small amounts.

Try to look at buffers from other companies. Eg. both EcoRi and HindIII cuts 100% in Buffer B from SuRE (Roche). It does not matter where you bought the enzymes.

Hope it helps

/M

increase digestion time or units number of Hind III will do the job. I prefer increase number of hind III units rathr that time due to the possibility of star activity.

To be sure i always do the digest in the appropriate buffer, then precipitate with ethanol and do the second digest. If buffers match but with reduced efficiency i add a bit more enzyme of the one that has the reduced activity.

I just use both REACT 2(Hind III) and REACT 3 (EcoR1) [invitrogen]. I hope that is okay!!

I did this double digest last week on PCR product for cloning.

I used EcoRI/HindIII in EcoRI buffer overnight. Nearly all my colonies had the insert in the proper direction.

-Matt