ligation transformation (View forum version)


Posted 11 October 2013 - 08:12 AM

hi guys,

you know when you use a enzyme to cut your self ligated vector background by choosing a site in your vector that is not there in your insert. hope you know what am talking about else tell me and i will elaborate.

when i add the cut enzyme, most people say just add a microlitre at end of ligation and incubate at optimum for around 10 minutes, and it should work, is that true? and also

well when you do that, do you need to heat inactivate the enzyme at the end of it? , if i do it might also affect the transformation?  can i just leave it and transform, will it inhibit tranformation?


Posted 11 October 2013 - 08:44 AM

thanks in advance..


Posted 11 October 2013 - 11:05 AM

That should usually work fine -- most enzymes will work in a ligase buffer. Heat killing will not usually be necessary, but it might help -- some say that heat killing a normal ligation reation improves things, but I've never done that experiment. Do not heat kill ligations with quick ligase buffer (PEG), which will lead to failure.


Posted 12 October 2013 - 03:18 AM

thanks phage..


Posted 12 October 2013 - 04:50 AM

I also think that most enzymes will work in ligase buffer. However, heat inactivation (plus salt removal, of course) is recommended for electroporation. For heat-shock transformation, it is not necessary to inactivate enzymes.