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It keeps getting better and better lol my ligase might be out to get me - (Aug/27/2008 )

Uhm.........when I started out doing this I didnt realize I could do so many things wrong at the same time........

just found out, through this forum that I need to inactivate my ligase after ligation for 10 min at 70°C

I had been reading protocols for ligation and such since I didnt get one from my supervisor and none of them mentioned this step, they mentioned a killer digest which I did and that was that.

So I ligated ON at 16°C and then added some Blg2 to the reaction and cut for half an hour.
Then transformed that. I have some colonies now which are being cultured as I write this

Ok im preparing myself to hear this......... what consequences can this lack of inactivation have?
Blg2 was also not heat inactivatable so I just left it there. I figured they couldnt possibly keep on working but I hope you can tell me why im wrong hahahaha

this is awesome hahaha

-nanu nana-

First, I've never done an inactivation of my ligase. I just transform after about 4 hours and leave the rest of the ligation on my bench overnight. If I don't have colonies I re-transform the ligation that sat out. I then throw the ligation out because I figure if I don't get colonies with overnight, it's not going to work and there's no reason to save the ligation. I am confused as to why you would be digesting your plasmid with BglII before transformation unless this is a digestion site that was removed from the MCS when your insert is ligated into the vector. This is a way to get rid of empty vector background. I really prefer to CIP treat the vector before ligation to eliminate background.


BglII is probably still active but it shouldn't have any effect on your transformation.

rkay - correct, that is why BglII was used - as a kill for an empty vector. I personally prefer kill sites to CIP/SAP.