Cloning using ds Oligos
Posted 22 March 2004 - 10:26 PM
I want to insert a small 30 bp sequence into a vector of mine using ds-oligo. I've created the oligos so that once annealed, they have the correct overhangs of the restriction sites.
As I'm waiting for my oligos, I had some questions... Does anyone have a protocol on this? Do I need 5' phosphates on both sides of the oligo? How do people prevent concatamer formation? What seems to be the optimum ratio of insert:vector?
Thanks for all your help!
Posted 23 March 2004 - 04:50 AM
I sometimes did the same thing, though a little different...
I designed my oligos with Endonuclease restriction sites at the ends - using enzymes that have a good "close to the end" cutting effinciency. So you get the phosphate automatically after restriction digest.
So, since you didn't digest, I'd advise you to either order the oligos phosphorylated or use Polynucleotide Kinase. Your vector should be dephosphorylated using Shrimp AP.
Formation of Concatemers can be checked in a high percentage agarose gel.
For everything else, we used standard clonig procedures (Vector/ Insert ratio of 1:3 molar)....
Posted 26 March 2004 - 12:52 PM
i always use T4 Polynucleotide Kinase to phospate the 5' end. it works well. and the vector should be dephosphorylated by CIAP or SAP(unless it was doulbe-digested).
for prevent concatamer formation, useing the correct ratio of the vector and insert(less than 1:8). before adding the ligation buffer and ligase, incubating the vector and insert mix at 65C for about 15 min, it will reduce unexpect ligation.