Generating "good" blunt ends

[Mick Jones]

Hi

I have always found that the repair of sonicated DNA to be very inefficient.  
Looking through the literature, possible solutions are the following.  They could 
all be used to generate random shotgun fragments for sequencing.  I haven't tried 
any of these methods, but I would trust them, even with my favorite teddy bear.

1.  Richard Gibbs (see Anal. Biochem., 210, pp 16-26 [1993] and 218, pp 300-308 
[1994]) treats first with mung bean nuclease to trim off single strand regions, 
then polishes with T4 DNA polymerase and Klenow.  It may be that the polymerases 
deal inefficiently with particular ss regions, and the mung bean (or Bal31) chops 
them off.

2.  In a protocol of Bruce Roe's, he kinases the blunt ended fragments with T4PNK 
after extraction of the desired size range from an agarose gel.  I've heard 
rumours, but not seen any data, that somehow the 5'-phosphate can be lost during 
the sonication/gel isolation.  Obviously such fragments will not ligate to a CIP 
treated vector.

3.  Richard Gibbs (see Anal. Biochem. 218, pp300-308 [1994]) uses an adaptor to 
ligate onto the ends of the blunt-DNA fragments.  The adaptor has an 11 base 
3'-overhang which cannot base pair to itself.  The M13 cloning vector has a 
complementary adaptor ligated to itself.  Actually, they generate the vector by PCR 
with uracil containing oligos, then UDG treat to generate the 3'-overhang.  
However, I think it is much simpler to ligate an adaptor to SmaI cut vector.  This 
prevents vector self-ligation, and also insert self-ligation.  In theory, you get 
one insert molecule in each recombinant.  By utilizing a long 11-12 base overhang 
you do not need to actual use T4 DNA ligase, the complementary ends will hold the 
recombinant together during the transfection procedure.  All you have to do is 
anneal insert and vector and add to competent cells.  This will overcome any 
inhibition of the ligase due to 'stuff' co-purifying from the agarose gel during 
the size selection of the DNA fragments.

By using all three you may increase the increase the number of recombinants.

I notice your control is AluI cut lambda DNA.  I also use it as well.  However, 
have you tried to put the AluI fragments through a mock cloning (i.e. polish, gl 
purify, etc.).  I'm sure you will get a reduction in cloning efficiency, and it 
might point to the step which is the problem.

I hope this makes sense.

Mick
_____________________________________________________________________
Mick Jones                      Tel: 081-740-3328 (+44-81-740-3328)
Department of Virology          FAX: 081-743-8331 (+44-81-743-8331)
RPMS,  Du Cane Road              Email: mjones at rpms.ac.uk 
London,  W12 0NN,  UK          URL: http://www.rpms.ac.uk/virology/virolhome.html
***********************************************************
"Smoke me a kipper, I'll be back for breakfast."   Ace Rimmer (Red Dwarf)
"Crackin' toast, Gromit."    Wallace (The Wrong Trousers)
_____________________________________________________________________