5 replies to this topic

[TDC_Tiger]

Hey,

So I see on several protocols that it is necessary to heat inactivate the T4 DNA Ligase. Can anybody tell me why this is important? I haven't been able to find a reliable answer anywhere so far.

Thanks,
TDC

[Curtis]

we don't heat inactivate DNA ligase and we transform the ligation reaction without inactivation. so far no issue. It must depend on application.However we heat inactivate other enzymes like T4 RNA ligase and RNase H during RACE experiments because the enzymes might disturb the next reaction.

[bob1]

TDC_Tiger, on 21 March 2012 - 06:40 AM, said:

So I see on several protocols that it is necessary to heat inactivate the T4 DNA Ligase. Can anybody tell me why this is important? I haven't been able to find a reliable answer anywhere so far.

In theory, the ligase will stay bound to the DNA and this MAY prevent/inhibit subsequent transformation - however like Curtis, I rarely heat kill the ligation and don't have any problems.

[TDC_Tiger]

Thanks a lot for the help. Good to know I wasn't doing anything too wrong with my protocol.

Cheers!!
TDC

[OA17]

Bob1 is right, the ligase binds to the DNA and this may prevent transformation, but as far as I know, it is more important to inactivate the ligase if you are performing electrotransformation (electroporation). In the case of heat-shock transformation, it is not so relevant.

[phage434]

It is important to heat kill the ligase if you intend to run the product on a gel.