Do you kill the ligation product? - (Jan/17/2006 )
I know so many questions at once but im setting a new ligation protocol and i will just die if it will not work out..... 
so please tell me if you kill the ligation and how do you clean after that? phenol chloroform is ok? dont mention kits because i think they dont have any here....
thanx a lot again....
I don't think you should need to? if there is a way to inactivate your ligase, it should tell you in the product literature (or if you don't save that crap it should be on the website)
however, I have never inactivated the ligase...I do not see a reason to do so...I just take the ligation out of the fridge and go directly to transformation (no clean-up in between is necessary either)
anyone else have a different theory?
I have heard from other lab friends that inactivating the ligase will increase transformation efficiency by about 2-fold which isn't that much. I don't know why that is the case, but inactivating only takes 10 mins by placing the tube in 65 degree heating block. I'll do some research to find out why inactivating ligase would improve transformation efficiency.....
thanx a lot but i what i am planning to do (some web source) is kill the ligase and then digest with some RE that is inbetween the insertion sides of my insert.....it is not in the insert but only in the vector...that way i will insure that only transformed colonies are got into E coli....what do you think?
I am not sure I understand...won't the restriction enzyme also cut recombinant plasmids?
thanx a lot but i what i am planning to do (some web source) is kill the ligase and then digest with some RE that is inbetween the insertion sides of my insert.....it is not in the insert but only in the vector...that way i will insure that only transformed colonies are got into E coli....what do you think?
ok ill explain by an example....for example i am ligating my insert by HindIII and EcorI.....in the vector between HindIII and ecoRi there is SalI site....so when i ligate new vector wont contain SalI site.....so when i digest ligation product with SalI all the unligated self ligated vectors will be linearized and will not be transformed....that is what i understood from the web is called killing the ligation.....
done that before. i cleaned it up by ethanol precipitation. worked pretty well too.
Generalyy I heat inactivate Ligase at 65C for 10' and go ahead for transformation. If you want to reduce the background colonies you can chose a RE site which closes upon successful ligation. Am not very sure if cleaning up this mix is very essential! But P:C extraction will definitely reduce the amount DNA you have.
sounds like a good approach..will have to try that one in the future. I love these forums!
thanx everyone ill try heat inactivation, RE digestion and then just precipitate the product, you are right i cant risk to lose any of my DNA ...