Religation again and again - Ligation problem (Apr/23/2007 )

Hello,
I am trying to ligate a 4kb insert to a 5,3kb vector.I cut them with KpnI and NotI, purify from gel and ligate with a ratio(vector/insert) 1:1 to 1:3.I do the transformation but I get no colonies.When I run 1ul from my ligation product cut with Kpn1 I take a band at 8kb only.I think that this is the insert religated but why dont it ligate with the vector?
Can you help me?
Thank you

Hi there!

1st, it can be sometimes hard to judge 8kb from 9kb, so maybe your ligation has worked...
2nd, KpnI and Not do nor produce compatible ends, so you may want to check if there are both sites really present in your construct and both only once....
3rd, you can always transform your vector/insert ligation into E.coli, then only ligation products containing the vector will show up, as the insert should lack an origin of replication and should therfor not be able to replicate....


mike

Hi,

No colony? Better check the vector resistant gene and then recheck the agar plate that you used, whether it's ampicillin or other kind of antibiotic, which should be compatible with the resistant gene present in the vector that you used.

Cheers

Thank you