cloning problem - sticky end cloning with PCR insert - (Jul/27/2005 )
My problem:
I have an insert 4 kbp, digested Xho1/not1.
I have a vector 5.5 kbp (bacpak8) digested and CIPed.
Ligation with different ratios, resulted in no colonies.
Why not?
DH5alpha and XL10 gold tried (both competent).
Also blunt ligation only resulted in a few colonies where there had been religation of vector.
In parallel I tried bluescript. Sticky ligation did not work here and with blunt ligation only a few colonies (I have not checked yet whether the insert is in).
Whats going on?
-hothevv-
How much extra bases do you have on your primers besides your restriction enzyme recognition sites? Most of them need more than the recommended 6.
You can try T/A cloning of your insert, cut it out of your vector, gel purify and then ligate.
-vairus-
QUOTE (vairus @ Jul 27 2005, 06:20 PM)
How much extra bases do you have on your primers besides your restriction enzyme recognition sites? Most of them need more than the recommended 6.
You can try T/A cloning of your insert, cut it out of your vector, gel purify and then ligate.
You can try T/A cloning of your insert, cut it out of your vector, gel purify and then ligate.
Thanks, For the Xho1 site there are only 5 before the cut, and for not1, 7bp.
I've heard about TA cloning, but dont have a protocol, do you know of a good one?
H.
-hothevv-
QUOTE (hothevv @ Jul 27 2005, 01:49 PM)
QUOTE (vairus @ Jul 27 2005, 06:20 PM)
How much extra bases do you have on your primers besides your restriction enzyme recognition sites? Most of them need more than the recommended 6.
You can try T/A cloning of your insert, cut it out of your vector, gel purify and then ligate.
You can try T/A cloning of your insert, cut it out of your vector, gel purify and then ligate.
Thanks, For the Xho1 site there are only 5 before the cut, and for not1, 7bp.
I've heard about TA cloning, but dont have a protocol, do you know of a good one?
H.
We use T-easy from promega and have always had good results. Make sure you used a Taq that adds the extra A at the end. Most do, and your catalog/supplier will be able to tell you for sure.
-beccaf22-