double digestion - double digestion (Apr/30/2005 )

Digestion using NdeI works well (I checked it out) but when I tried the second digestion with XbaI, DNA simply disappeared!!! Does anyone can help me?

hi
ok, i assume if you do a sequential digestion you precipitate the DNA after the first one. So i suppose you loose your dna after precipitation (maybe pipetting it when removing ethanol, or during drying, i've noticed that dna can just go in the air!!)

according to NEB, the double digest is possible. (NEB buffer 2 + BSA)SO i would recommend you to do the double digetst.

fred

Neb buffer 2 composition is available here.

Thanks for answering Fred. But there´s a problem: I can´t use Nde and Xba together for the same digestion (although the same NEB can be used for both) ´cause I need to repair DNA after the first digestion (with Nde). I´ll try to precipitate DNA after reparation. I wonder it works well.

Angelica