Blunt end ligation debate.

Darren Natale camdna at ubvms.cc.buffalo.edu
Wed Jul 14 11:19:00 EST 1993


In article <1993Jul14.085421.25969 at aristo.tau.ac.il>, pc386 at ccsg.tau.ac.il (BENNY SHOMER 9238) writes...
>Hello Bionetters.

[stuff deleted]

>Following transformation, it was clearly evident that the CIP reaction failed
>to perform, as I obtained many colonies in the control plate of Blunted/CIPped
>self-ligated vector.
> 
>Now my debate: Should I "go for it" anyway, and try to pick up positive coloniesanyway, regardless of the fact that the CIP reaction failed, or should I insist
>on performing a more efficient CIP, and discard the colonies I already have.

[more stuff deleted]

I am a firm believer in the statement that the most time-consuming step of
cloning is the diagnostic step, i.e. finding the clone you want. For this
reason, I always try to limit the number of possible transformable products
after a ligation reaction. When CIP fails (as it often has for me), and gel
purification seems like overkill, I do a restriction digest after the
ligation reaction to remove unwanted products. The enzyme used will depend
on the type of ligation of course. For instance, if your vector was digested
with an enzyme that leaves blunt ends, and your insert has blunt ends from
a different enzyme, then you could digest the ligation products with the
enzyme used to prepare the vector. This assumes that such a digest would not
cleave the insert or the vector/insert junction. If you have any of your
ligation mix left over, this might be worth a try. This same method can be
used on your colonies as well--simply pour some growth medium on the plate,
tranfer the liquid to a tube, grow for a while, miniprep and digest the
products with the necessary enzyme. Use the digested DNA for another
transformation. I only do this if I have neither ligation mix leftover nor
starting DNA left.

On a side note, I have also digested certain insert DNA with an enzyme that
leaves a single base overhang (e.g. Tth 111 I or Bst NI) prior to ligation.
Such products are for the most part unligatable.

D. Natale



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