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Problem with cloning RAPD fragment


Fri Jan 15 21:30:35 EST 1993


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



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