pvu I ligation problems

TFitzwater at Gilead.com TFitzwater at Gilead.com
Thu Apr 12 18:12:37 EST 2001


>Daniel Mebrahtu (danielmeb at hotmail.com)
>Wed 11 Apr 2001 - 00:16:32 BST
>dear members,
>i am having a problem ligating Pvu I ends in a very important project i
>started recently. can anyone help me out if she/he have had a similar
>problem and triumphed in the end.
>daniel

Daniel,


I have seen this problem before (many years ago), and later ran across a
reference that the Pvu I ends need to come from a dam minus strain of E.
coli if you want to ligate these ends.  You can cut dam plus DNA with Pvu
I, but you can't religate it.  This is not something the restriction enzyme
suppliers mention.  Both the vector and the insert need to be isolated from
dam minus E. coli.  (The reference is at home, so I can't tell you what it
was right now.)
Since I didn't know about the methylation problem at the time we were
having difficulty, I told the graduate student to try some of the New
England Biolab's high concentration T4 DNA ligase (2,000,000 NEB units/mL
= 30,000 Weiss units/mL) we had in the freezer to see if we could force
ligation.  I don't recall at this time how many units he used in the
ligation reaction.  He got ligation to occur and isolated colonies.
Subsequent sequencing revealed that while the insert was correct, the Pvu I
site was missing, presumably due to exonuclease contamination of the Pvu I.
Since we didn't care about the Pvu I site in the final product, this was
good enough for us.

Tim Fitzwater
Associate Scientist
Gilead Sciences

---




More information about the Methods mailing list