Molecular cloning of SalI/PacI digested product in modified pBluescript backbone - (Aug/16/2006 )

Dear Frenz,
I am having problem in cloning PCR fragments sequentially digested with PacI/SalI respectivel in pBluescript backbone. Both enzymes are from NEB and don't have compatible buffers so the digestion was carried out for 4 hours for PacI (Buffer1) and then overnight wiht SalI (in buffer 3). Ligation was carried out at 4ºC overnight. With the faliure of this strategy I tried cloning this fragment in pGEMT to make sure that both the enzymes cut well and elute out the respective product wiht SalI and PacI sticky ends. But again this fragment cannot be ligated to the pBluescript backbobne. So please help me out, this is a crucial clonign for me. Is PacI/ SalI cloning a prob lem in general? Is there any trick for such cloning which doesn't look complicated at all.

SalI digest can b a bit tricky at times. I would digest with PacI and then with SalI for both the vector and ur PCR fragment.

Also look at other posts in the forum for help with salI digest.

Good Luck !!!

Hey Malik,
Then how is life in Washington? Thanks for the sugeestion. I have already tried out first PAcI and then SalI digest. But it still doesn't work, then what else you can think of. I have even clone d my PCR fragment in pGEMT. inorder to be sure about the digest.

Cheers
Amit

hi amit,
u may also want to be sure about your double digested vector? salI digestions as malik said are generally tricky. if u have any other fragment cloned into pKS vector and if PacI/salI restriction sites are still present in that recombinant vector, u may cut that recombinant vector with both enzymes and you will be sure that double digestion is not the problem in your cloning experiment.

u may also try to alter ligation reaction conditions. molar ratio of ends, time, temp!

Hi Amit,

i would suggest to reduce digestion time from 4 hrs to 2hr or even an hour for Pac1 and for sal1 also 2 hrs.

Also try ligating at RT with T4 DNA ligase.

Good Luck !!!