Religation with single digest vector - (Aug/09/2006 )
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.
what are your ligation conditions? 4C, 16C or RT?
o.n. 14 ºC
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!
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!