Ligation of two oligos into a vector - (Jul/01/2005 )
Hi all,
I am getting frustrated, I don't know what to do.
I have two oligos nonphosphorylated, one with blunt end, the other with sticky end. The insert was cut with BamH1/fill end/cut with EcoRI. I did the ligation with oligos, one should go at the blunt end, the other at EcoRI end. The size of the vector is 0.69 kb, plus oligos should be 0.718bp. I checked on 1.5 % gel. Seems to be fine, but i am not really sure. Then i have to ligate this into other vector cut with another two enzymes.
No colonies. The vector is fine. I am not sure about the insert plus oligos. How to control if I have the right oligos in the right place? I can not do PCR, is a big difference between Tm. Just to cut with the restriction enzymes maybe,I didn't try this, because I don't have enogh insert.
Should I phosphorylate the oligos before the first ligation?
Thanks a lot for your help,
hi
seems it's essential to phosphorylate your oligos...
seems it's essential to phosphorylate your oligos...
Hi thanks a lot for your reply. But the insert is with both end phosphorylated, I cut with EcoRI at one end, and should be fine. At the other end I cut with BamH1 and then I filled with Klenow. Should be phosphorylated also? I am not sure for this end but Klenow enzyme should add dNTPs, and also keep the end phosphorylated.
Actually I did an exp. insert ligated with one oligos, for EcoRI end, I got different bands. My conclusion was that the blunt end of the insert could ligate with other insert blunt end.
Am I right?
Maybe the transformation was wrong, I didn't keep the plates at 37 degree for 1 hour, just at room temp 30 min, which was 20-21 degree.
thanks a lot
hi
actually yes.blunt ends are copmpatible. But in a standard ligation, even if you have two differnt restriction sites at ends of your insert, there is a compatibility too. And ligation of 1 fragment with plasmid occurs generally. Hence i don't think ligation of blunt ends the source of your problem. Increase the amount of insert if you can. For the first end, cleaved by EcoRI, it's phopshorylated. By klenow, i'don't know...but i believe that klenow add nucleotides for 3' end and therefore does not affect 5' end.
Maybe the end filled by klenow do not have a 5' P ? 
Try phosphorylation and please tell me the results.
fred
actually yes.blunt ends are copmpatible. But in a standard ligation, even if you have two differnt restriction sites at ends of your insert, there is a compatibility too. And ligation of 1 fragment with plasmid occurs generally. Hence i don't think ligation of blunt ends the source of your problem. Increase the amount of insert if you can. For the first end, cleaved by EcoRI, it's phopshorylated. By klenow, i'don't know...but i believe that klenow add nucleotides for 3' end and therefore does not affect 5' end.
Maybe the end filled by klenow do not have a 5' P ?
Try phosphorylation and please tell me the results.
fred
Hi Fred,
Thanks again for your advice. I am sure that the insert should be OK. But for the second ligation with the new inser + vector I think now there are problems, because my insert is complete dephosphorylated.
I have actually the vector with 5' P, but the insert without 5'P (because the ends of the insert are know the ends of the oligos with restriction sites compatible with the vector but without P.
Normally the ligation could be OK, and the 5' from the oligos could be nicked to 3' of the vector in bacteria.
The DH5alphacells from Invitrogen are able to do this? I don't know. I'll try to phosphorylate the insert, I don't know how much I have left.
thanks again