Generating "good" blunt ends
mjones at rpms.ac.uk
Wed Nov 22 06:06:34 EST 1995
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  and 218, pp 300-308
) 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
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
3. Richard Gibbs (see Anal. Biochem. 218, pp300-308 ) 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 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
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