Ligation problems

P.C. PC at no.email.sorry
Wed Feb 18 19:23:42 EST 2004


that happens only with this pair of primers?

I had it once - a nightmare, four weeks were spent: PCR was just great, 
but nothing further worked. At the end I gave up on the restriction and 
tried to clone the fragment into a Topo vector - even that did not work 
well. I finally got a couple clones, each of them had a part of the good 
sequence (same thing - deletions at the ends) - from these two I 
assambled the complete cDNA. After that all other primers on the same 
cDNA worked well, it was re-cloned by now into at least a dozen vectors...

my explanation - something was wrong with the oligos - not completely 
deprotected may be? Funny thing is that the ligation worked - I could 
see the ladder of the insert self-ligation. As if the DNA did not 
replicate in E. coli....  Deprotection is simple (but stinky) - just 
heat the oligo in ammonia for a while - you probbably know it better, as 
you make the oligos yourself.

best,

Peter

Paul Shinn wrote:

>    We have run into some problems with our ligations.  Our transformants 
> are either non-existent, non-abundant, incorrectly cloned, or vector 
> only.
> 
>    We do directional cloning of PCR products using Sfi I and all our
> transformants are sequence verified by us.  The vector has 2 Sfi sites 
> and is digested with Sfi I and then gel purified.  Because of how Sfi
> cuts, there should be no self ligation of the vector and the PCR products 
> (ends are also digested with Sfi) should clone in only one direction.
> 
> GGCCN NNN|NGGCC
> CCGGN|NNN NCCGG
> 
> But like I said, we are getting vector only clones or the transformants
> have backward inserts or there is truncation of the PCR product ends
> during the ligation.  And sometimes (rarely) they actually work.  Most of
> the clones look like they are blunt end ligated rather than sticky end
> because the Sfi site is gone.  Sometimes the Sfi is gone on the insert
> side and sometimes on the vector side.  There is no rhyme or reason to
> this.
> 
> For the most part the PCR is using Taq.  Then sodium acetate/ethanol 
> precipitated.  Digested with Sfi overnight.  Precipitated again and then 
> ligated overnight at 16.  For larger inserts we use a proof reader and 
> then do phenol/chloroform and then sodium acetate precip.  We had some 
> problems with the proof reader chewing back the ends of the insert.
> 
> I synthesize the PCR primers for cloning.  The forward primer is 56mer; 
> the reverse primer is 43mer.  If the oligos are degraded, the 3' end would 
> usually go first, right?  If so, the restriction sites at the 5' end 
> should be intact.  PCRs are still working, too.
> 
> Each person has their own stock of vector and generates their own clones.  
> It's hard to believe we're all doing something wrong at the same time.  
> I worry that we're seeing the result of multiple problems like vector is 
> degraded and PCR ends are degraded independently.  Can a sticky end blunt
> ligate to a blunt end?
> 
> We're out of ideas.  Thanks for your help.
> 
> Paul




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