Problem cloning RAPD PCR fragment

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Sat Jan 16 15:49:00 EST 1993



%%%%%%%%%-Yin Chai writes-%%%%%%%%%%%%%%%%%%----
Hi, 
        Has anyone successfully cloned a fragment from RAPD PCR? I have not
been
able to do it.

        I have been trying to clone a 2200 bp fragment from a RAPD PCR of
mouse
DNA (using a single 12 bp primer). I ran out the PCR products on a 1%
agarose/TBE gel. (Type of agarose don't seem to matter. I have tried
SeaPlague, SeaKem, and BRL.) I then cut out the band, ligate it to
Promega's pGEM-T vector, and transform BRL's DH5alpha (no white colonies)
or Promega's JM109 (24 white colonies max). A miniprep of these whites
revealed 18 false positives (no insert) and 6 inserts that are smaller than
the band I cut out (700 bp to 1300 bp). I have tried many variations of the
above protocol (in gel ligation, NACS purification of the band,
electroelution, plain phenol choloroform purification of the band etc.) and
24 white colonies is the best so far, and I have never got any insert
bigger than 1500 bp. 

        Any ideas on what's going on? Someone suggeted homologous
recombination. I
am trying Stratagene's SURE cell (recA, recB, recC and recJ minus). Any
other ideas? Thanks.

Yin-Chai
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
 HI Yin-Chai
    I wonder if you phosphorylated your primers before
   PCR or the product after?  The Oligos you get from 
the synthesizer do not necessarily have phosphates unless 
you order them that way.  If you are phosphatazing your vector (with CIP)
you will never get any clones unless you kinase your product or start
with primers that have been phosphorylated.  The Best way to do this 
kind of cloning is to CIP your vector ( remove the phosphates) and have
phosphates on the product.  This way the only product you get is due to 
 an inter-molecular ligation.  The Blue:White technique is ok for sticky end
clonings but I've found it does not work that well with blunt ends.( a lot of 
false
positives and UCOs [unidentified cloning objects])
 I presume you  are cloning this fragment as a blunt end.  There are a couple 
of
tricks I have that help the blunt end cloning of any fragment including PCR 
products.
This includes a method of band purification from agarose which I recently
eluded to in this forum. Let me know if your interested.

Dan Gietz 
U of Manitoba
GIETZ at BLDGHSC.LAN1.UMANITOBA.CA



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