Triple ligation with IRES - (Jun/01/2006 )
getting a bit frustrated with a triple ligation that doesn't seem to work (i.e. very few colonies, none with insert, very low background = religated vector) I was wondering if anybody has any advice concerning the issue:
1) any suggestions for insert:insert: vector ratio? Would that depend on the size of the inserts?
2) one of the 2 inserts is an IRES element - does that require special considerations in triple ligations?
3) has anyone compared the Rapid ligation kit from Roche with the regular T4 ligase from NEB?
Any comment appreciated!!!
We routinely do triple ligations. The correct ratio is 1:1:1 molar ratio, so yes, it depends critically on the lengths of the fragments. Ligase doesn't know what it is ligating, so it knows nothing about IRES elements. If you are doing sticky end ligations, then just use T4 ligase. For blunt end, use the rapid ligation kits. How are you preparing your inserts? UV damage is a common problem if you are cutting from a gel. Low ligation concentrations of DNA will favor your desirable product over concatamers. How are you transforming? Perhaps you have low transformation efficiency rather than low background?
We routinely do heat shock transformation, our bacteria give around 10^7 cfu/ug, which I think is ok.