Cloning Help!! - It is getting very annoying... (Jul/29/2010 )
cluelessstudent on Sat Jul 31 10:32:14 2010 said:
it's funny tho, many of my lab workers said that without cip, the sticky ends somehow get back together even when they are non-compatible with 1 another... any1 know why?
Well, my first response -- no offense -- is that "many of
-HomeBrew-
Ok, this week I tried cloning without CIP - massive amount of colonies in the negative... compared to cip... wtf is going on?
-cluelessstudent-
HomeBrew on Sat Jul 31 14:03:42 2010 said:
cluelessstudent on Sat Jul 31 10:32:14 2010 said:
it's funny tho, many of my lab workers said that without cip, the sticky ends somehow get back together even when they are non-compatible with 1 another... any1 know why?
Well, my first response -- no offense -- is that "many of
It happened to me twice when I tried to ligate an insert to my vector by AflIII / SacI sites (they are non-compatible) and I got the sequence having these sites ligated to each other, even though I treated CIP the vector. Just one of many cases I observed that the theory is not always true.
-Quasimondo-
The fact that they ligated together was probably *caused* by the CIP (damaged ends) rather than mitigated by it. Do you thus CIP everything, regardless of enzyme overhang combatibility? Madness, I say...!
-HomeBrew-