CIP of Vector and Kinase of Insert? - Is it necessary in blunt end cloning? (Nov/30/2006 )
I am in a big trouble to clone the gene cut with AscI and EcoRI and then filled the end with T4 polymerase. The vector was cut with the unfortunate SalI and then T4 treatment and CIP.
Ligastion was set and the transformation.
All the colonies are showing the same fragments when digested with NcoI and no difference in orientation? The size of the fragments are not matching the total size of inser+vector.
How are the things going I don't know? I had tried it many times and no positive clones are seen
Is it must to perform the CIP of vector and kinase of insert in blunt end cloning?
I did all the things for 5 times (150 minipreps), NO RESULTS.
Hope to get the help soon.
Thanks in advance!!!
well CIP for blunt end is necessary.
see the post of kathy for the kination of insert :
your end is obtained after restriction --> no need to phosphorylate
in other case --> phosphorylate