Three fragment ligation

David C. Logan david.logan at plant-sciences.oxford.ac.uk
Fri Jan 17 12:31:28 EST 1997


Andrew Doherty wrote:

>Hi everyone

>There must be lots of people out there ligating three bits of DNA
>together instead of just two - but I'm having major problems with it.
>I'm constructing a fusion protein, but have very few restriction sites
>to choose from which I can use, hence the need for the three fragments.
>My vector is pcDNA1/amp (dephosphroylated) cut with NotI/SphI, my
>receptor cut with NotI/HindIII and GFP cut with HindIII/SphI. So 
>putting all three together should give me a circular plasmid. The 
>ligation looks fine on a gel - the GFP (800bp) and receptor (3Kb) both 
>disappear and I get a ladder of products. But no colonies!! I know that 
>the bugs are fine, as other transformations are working well - so my 
>question is, are there any tricks in getting this sort of ligation to 
>work or is it just luck??!!@# I'm using BM's T4 ligase at 4 deg C 
>overnight.

I have just performed the same kinda thing, in my case a signal seq. cut 
with NcoI/SpeI to GFP cut with SpeI/BamHI into pRTL2 cut with NcoI and 
BamHI. Not really knowing what ratios to use a did a 3:3:1 ratio of ends 
(i:i:v, 50ng vector) and ligated o/n at 16 deg C with GIBCO T4 ligase. I 
then transformed good CaCl cells with the whole 20ul ligation mix. I got 
loads of colonies but only one fifth or so were correct insertions 
(restriction digests of minipreps with NcoI/BamHI or all three enzymes. 
Unfortunately this is the only time I have tried this and actually 
thought it might fail since everybody I talked to about a tripartite 
ligation sucked in breath and said "dodgy"! As a back up I had ligated 
both inserts into separate plasmids so that I could be sure they were 
cut (PCRed fragments using in-primer RE sites meaning that I could not 
actually see a size diff. to prove fidelity of cutting) and was going to 
ligate the inserts first and then into the plasmid but luckily I didn't 
need to. Sorry I can't give any particular tips - I guess I'd vote for 
the luck option,

I hope yours changes

David
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david.logan at plants.ox.ac.uk

David C. Logan
Department of Plant Science
University of Oxford
South Parks Road
Oxford
OX1 3RB

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