Religation with single digest vector - (Aug/09/2006 )

Dear all,

I am struggling with a simple problem. I can not religate a simple single digest of the plasmid pYX213, digested with EcoRI. As I could not get any clone in the vector, I decided to make the control of single digest religation, and nothing.

I guess I have made all the controls possible
I changed ligase (NEB),
I changed buffer,
I used other buffers from other suppliers,
I used DH5, DH10, TOP10, TG1 E.coli competent cells
I cut with other enzymes and no religation

I digest 2 hours, gel purify the band, check that I still have product on another gel.
My ligation reaction contains at least 100 ng plasmid, 5 U, 2h, RT,. Transform 5 ul with 50 ul cells.
And nothing. I even made the test with other plasmids, and the worse happened, nothing. Only a brand new plasmid recently bought to Invitrogen religated (even though not very efficienly, but better than o colony!!). I am afraid something is wrong with all my stocks, but don´t know what.

Any suggestions?
Thank you.

Aristote

what are your ligation conditions? 4C, 16C or RT?

I tried
2h RT
o.n. 14 ºC
o.n. 4ºC
and nothing.

In which buffer is the DNA, the cut one ? Try changing the buffer (tris or TE) to water.

I already tried to resuspend in water, TAE and TE. Always nothing.

well avoid TE, and try resuspend in 10mM tris ph8-8.5 (elution buffer of most kits)
did you try to avoid gel purification step?
i know this may be a silly question regarding background possibilities, but i face problems of ligation when gel purifying...

no, I always gel purified my bands!
But never tried Tris alone. Let´s give it a try!
Thanks

extract the plasmid again,and digest it with another enzyme of MCS.if there is still nothing,you 'd better change your enzyme,maybe your enzyme is expired or stock at inproper condition.Good luck!