To inactivate or not to inactivate ligase before transformation? - (Dec/12/2005 )
I'm trying desperately to clone a XmaI/BglII cut fragment into the pGL3 basic vector (also cut with XmaI/BglII). Both fragments have been purified from gel and checked on gel as far as possible. I've tried Quick ligase (NEB; 5 min at RT) and "ordinary" T4 ligase (NEB) at both 4oC and 16oC for up to 48 hours. But no colonies. The vector does not religate confirming that both enzymes have cut (I'm only able to visualise the effect of the first enzyme I use, which has been XmaI; then I purify cut from uncut vector after gel electrophoresis before using the second enzyme followed by purification on column). The insert has been "pre-cloned" into a pCR 2.1 TOPO-vector and then isolated from gel after XmaI/BglII cut and purified on column. Everything looks OK, but no colonies... Uncut pGL3 gives plenty of colonies.
I'm running out of things to modify, but my question now is: is it necessary to heat inactivate the ligation mix before transforming the bacteria? I use heat-shock competent cells. If heat inactivation will most likely increase the transformation efficiency, can I use my 48 h lig-mixes that I've stored at -20oC since last week or should I start all over again?
Also: does anyone know if it makes a difference if I use DH5a or JM109 bacteria for this?
What molar ratio's of vector to insert have you tried for your ligations? How much of your ligation mixture are your transforming and how big is the volume of cells you're transforming into? Have you tried ligating uncut vector and transform this (to see the possible effect of some compounds in your ligation mixture on your transformation)?
I've done heat-shock transformation of inactivated and non-inactivated ligations and both have worked (don't remember which one gave more colonies, but after 48h I don't think you will still have a lot of ligase activity left).
I don't know about DH5a of JM109 being better or not. Use the ones with the highest transformation efficiency according to the manufacturer. If necessary, transform isolated plasmid into the other strain.
i know that DH5alpha cells have better tranformation efficiency.
for ligation inactivation i do it for chemically competent cells.
you may try for enhancing ligation : pre heat the cutted vector 65° 5' and let it cool on ice. add buffer and insert and enzyme.
i would try with your -20° stored lig mix.
Our experiments show a 10x to 100x reduced colony count on heat inactivation of ligase in the NEB quick ligation kit; this agrees with their web page comments.
I've noticed that the Quick ligase should not be inactivated so your data confirms this in a very nice way; thanks
But what about the "ordinary" T4 ligase from NEB (not the Quick ligase); it can be inactivated at 65oC for 10 min but has anyone done any tests comparing with/without this inactivation? Fred_33 does this inactivation when using chemically competent cells, but I'm curious if this could be expected to have a significant influence on the colony counts or not.
Hey Sonjaa.. I have not done a side by side comparison of heat inactivated vs not.
But I have always done 70C for 10 min followed by a quench on ice and quick spin for my ligations.
Try a room temp ligation for about 4 hrs also instead of 16C. That may help.