adjacent restriction sites - (Jun/03/2009 )

Hi Colleagues,
I am trying to clone a PCR product /BglII/NotI into a TMR vector which has these sites adjacent to each other. I do not have any other choice but to use these enzymes. Can someone help me to get a successful ligation? Will sequential digestion work?

PCR your vector with overhangs that add additional bases to the cloning site, then cut and ligate. The vector is a piece of DNA, not some magic thing that the ancients handed down to you. You have control.

Unfortunately the vector is hand drawn and the sequence is not published, it will be rather difficult to design primers against an unknown sequence
nevertheless its a good idea , thank you for your help

ntlfly on Jun 4 2009, 04:16 AM said:

I'm a bit confused with your question. You say you want to ligate with BglII/NotI, and then you say you dont know the sequences? well, if you want to digest and ligate with those RE that's your sequence, which you can get from any RE manufacturer website.
Here they are from NEB (the / sign indicates where the enzyme cuts).

BglII
5'---A/GATCT---3'
3'---TCTAG/A---5'

NotI
5'---GC/GGCCGC---3'
3'---CGCCGG/CG---5'

So, design primers with those flanking sequences (remember to add at least 3bp at each 5'end), PCR, digest you product, digest your vector, ligate... in theory is that straight forward (in practice problems might start coming up).

There's always another choice, why not just use either one of the sites and do a blunt-end cloning?

what really matters is: how near are they?

ntlfly on Jun 4 2009, 08:57 AM said:

ntlfly on Jun 3 2009, 05:57 PM said:

i have had to do a similar cloning with a difference of 6 bp between the 2 RE sites. i performed sequential digestion for my vector n it worked w/o any prob.