three part ligation with sticky ends not working! - (Mar/09/2012 )

I cannot get my ligation reaction to work. I have 3 part ligation with sticky ends (sizes roughly - insert1 2000bp, insert2 1400bp, vector 7000bp). I tried T4Invitrogen ligase (1h at RT) and Quick NEB ligase (5-10min at RT). For transformation I use ultracompetent XL-10Gold cells (Stratagene).

I perform double digestions for all three plasmids:
1)Vector: KpnI HF + XbaI in buffer4 1h at 37C, then add phosphatase and incubate 15min at 37C and then incubate 20min at 65C to inactivate phosphatase
2)Insert1: KpnI HF + DraIII in buffer1 1h at 37C
3)Insert2: XbaI + DraIII in buffer3 (75%activity of XbaI) 1h at 37C

Then I run digested mixes on agarose gel and cut out the bands of interest (I can see that vectors for Insert1 and Insert2 are nicely digested, but for vector I cannot say since the cut part is very small – it runs almost the same as undigested vector).
I purify the bands of interest using Qiagene Kit and then perform ligation reaction using ratio 1:3:3 (vector:insert1:insert2).
I also tried ligation without gel purification of bands (so I don’t expose DNA to UV light), in which case I just purify digestion mixes using DNA purification Kit (Qiagene).
I always get very small number of colonies, run colony PCR and get inconclusive results (for PCR for insert1 I don’t get anything, for PCR for insert2 I get a band of interest and unspecific bands). I send it for sequencing and get nothing!
Does anyone have any advice re three part ligations? Is it much more complicated than 2 part ligation?

You should not need sequencing to tell if you have good ligations. You can simply miniprep and examine the length of the insert. I don't see anything wrong with your approach, but I would use a 1:1:1 ratio (this is unlikely to be the problem). I would guess the main problem would be background from uncut or partially cut vector, and would expect vector only colonies to dominate.

phage434 on Fri Mar 9 15:20:14 2012 said: