3 replies to this topic

[netnus]

HI,

I am using BamH1 and Nde1 to double digest the PCR product and Vector.The BamH1 and Nde1 are from NEB. I try using NEBuffer2 as buffer to digest,but the cleavege is poor.
Anyone has ever used these two REs together for double digestion? Which buffer do you use? Any tricks to increase the the efficency?


Thanks!

netnus

Edited by netnus, 02 December 2004 - 08:12 AM.

[Janina]

Try following link:
http://www.fermentas...gest/index.html

It is from MBI Fermentas but you can also see how fine they work together. Perhaps you can also compare buffer condition.

[praveen_iitb]

HI,
Many times I also observed incomplete Double digestions. The problem is not only with BamH1 and Nde1(many other combinations also). Using commen buffer is always problematic.
To avoid such situation
1) do single digestion at a time with the appropirate buffer.
2) digest 1st, with the enzyme required low salt buffer and then with which required high salt buffer. do phenol chloroform extraction after each digestion.
3) check the concentration of plasmid being digested( too much plasmid will give u incomplete digestion).
Or check ur plasmid preparation it may be contamined with proteins.

try this
praveen

[zhgljj1998]

HI,
 
  I am using BamH1 and Nde1 to double digest the PCR product and Vector.The BamH1 and Nde1 are from NEB. I try using NEBuffer2 as buffer to digest,but the cleavege is poor.
Anyone has ever used these two REs together for double digestion? Which buffer do you use? Any tricks to increase the the efficency?


Thanks!

netnus

do not use buffer2 please. i had been troubled for two month. try to use other buffer. good luck