12.4kb vector 6.7kb insert - (May/12/2012 )
I am having problems ligating a 12.4 kb vector and a 6.7 kb insert.
I cut them in 50 microliter volume with HF Restriction enzymes (I do not overdigest), AP the vector with antartic phosphatase, and then dna purify with the quiagen columns. I do admit I exposed the DNA to UV light, but less than 1 minute, run a little aliquot to check that it was a single band and I did, used 10 ng/microliter concentration in a 10microliter volume ligation (ratio 1:1/1:2/1:3), ligation 4C overnight, DH5 alpha/TOP10/XL2 blue cells, have few colonies with the backbone AP and 4x more in the ligation plates (after several trials decided to go with DH5alfa which gave me more colonies than the others).
Since in the vector there is pBR322 with amp resistance gene (and eventually would love to cut off that one) and in the insert there is kan resistance gene in a high copy backbone, I plated the chemically transformed cells in amp plates (100u/ml) and those in kan plates (50u/ml) but nothing grew in the kan plates (some bacteria are there trying to grow...).
therefore I started some colonies for each ratio in kan broth 50% and soc media 50% and some one grew... but none of the colonies minipreped so far are the one I am looking for,..
Any idea on how should I proceed?
thank you very much.
val80 on Sat May 12 21:54:31 2012 said:
Since in the vector there is pBR322 with amp resistance gene...and in the insert there is kan resistance gene in a high copy backbone...
What do you mean by saying the insert is high copy? Is the insert plasmid-derived? If it is, and it contains a plasmid origin of replication and is in the same incompatibility group as pBR322, the cloning will not work without one of the ori's becoming inoperable (i.e. mutant).
Thank you so much!!!!!!!!!