Double digestion/ligation problem - (Aug/25/2005 )
Hi,
I have been trying to clone a 1500bp insert into pET3a vector. since i have a Nde1 site inside my insert i am digesting my insert with Ase! and BamH1. if i do a double digest for more than 1 hour i get multiple bands. also after i digest both the vector (BamH1/Nde1) and the insert (BamH1/Ase!) for 1 hour, gel purify, ligate and try to transform i am unable to get any colonies.
ANY SUGGESTION?
debi
hi,
i wonder if Ase1 and Nde1 give u same overhang, if not, then there won't be any ligation, for Ase1+BamHI, u can try sequential digest or digest in NEB buffer3, with double BamHI, for 2 h, for more specific information on Ase1, call the company that sold u these enzymes.
good luck
nbj
i wonder if Ase1 and Nde1 give u same overhang, if not, then there won't be any ligation, for Ase1+BamHI, u can try sequential digest or digest in NEB buffer3, with double BamHI, for 2 h, for more specific information on Ase1, call the company that sold u these enzymes.
good luck
nbj
yes, Ase1 and Nde1 give same overhang. when using double digest (BamH1 & Ase1) i use NEB Buffer2. i am trying sequential digestion. i will also try with Buffer3 as suggested by you. thanks a lot.
debi
  i wonder if Ase1 and Nde1 give u same overhang, if not, then there won't be any ligation, for Ase1+BamHI, u can try sequential digest or digest in NEB buffer3, with double BamHI, for 2 h, for more specific information on Ase1, call the company that sold u these enzymes.
good luck
nbj
yes, Ase1 and Nde1 give same overhang. when using double digest (BamH1 & Ase1) i use NEB Buffer2. i am trying sequential digestion. i will also try with Buffer3 as suggested by you. thanks a lot.
debi
hi...
try adding 1X BSA while doing double digestions as BSA would not allow any non-specific digestion.
goodluck
Anjali
anjali,
i always use 1x BSA in my digestion recipe. any other suggestion from anybody.
thanks
debi