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Problem with cloning RAPD fragment

Miao, Zhonghe miao at OCELOT.RUTGERS.EDU
Sat Jan 16 23:26:08 EST 1993


Dear Yin-Chai:
  I think there are two problems in your question.  First is how homogenous
your amplified band is.  Is it a single species of DNA fragment? If you have
evidence that the band is essentially a single fragment, you may do a brief
restriction mapping of the amplified products.  As a matter of fact this can
tell if the band is a single species of DNA fragment.  The second problem is
that of DNA cloning.  You may want to treat the isolated frament with T4 DNA
polymerase to flush it so that it can be cloned into either SmaI or EcoRV site
of SKII vector or some other vectors.  This cloning method should not be
different from a routine DNA cloning technique, regardless the source of DNA. 
However, blunt end ligation for a 2 kb fragment may not be as efficient as what
is described in manual books.  Therefore I suggest you use the following
strategy to either clone full-length or part of the fragment.  Based on the
restriction sites you will have about the fragment, cut it with one ortwo
appropriate restriction enzyme(s) so that two compatible cohesive termini to
the sites in the polylinker region of the vector can be created.  You may have
a good chance to find such two sites in your fragment, as there are so many
sites available in polylinker region of SK or pUC vectors.  Good luck with your
cloning!

Zhonghe Miao
Waksman Institute
Rutgers University (908-932-4296)
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To: arabidopsis at net.bio.net
From: Yin-Chai CHEAH on Fri, Jan 15, 1993 10:33 PM
Subject: Problem with cloning RAPD fragment
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Date: 16 Jan 93 02:30:35 GMT
From: (Yin-Chai CHEAH)
Subject: Problem with cloning RAPD fragment
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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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