Cloning 12-15kb DNA

Winoto lab lab_winoto at
Mon Jun 27 16:01:29 EST 1994

In article <1994Jun27.163249.1 at>
gritsun at writes:

> I am working with a 12-15kb DNA molecule which has not been stable in vectors 
> pGEM3, pGEM4 and pBR322. I'd be grateful for any suggestions for a suitable
> vector.

The problem is most likely not with your vector but rather with the
bacterial strain that you are using.  Check to make sure you are using
recA- strains, genotypes of the strain you are using are given in the
reference sections of the GIBCO-BRL and NEB catalogs.  If the strain is
okay and you still think the vector is the main problem try cloning
into pSP72 (promega). I have routinely cloned 10-16 kb fragments into
this vector without much of a problem and have made constructs with up
to 27kb by inserting 5-7 kb fragments step by step, but there is really
nothing apparently special about the plasmid that I can tell.  In some
cases, however, when I've  had a particularly low frequency of "insert
intact" transformants I've used the colony hybridization method to
obtain the few transformants that have the insert that I wanted.  Best
results with this method involve the use of "colony screen" filters
(Dupont).  Prior to using this method isolate a piece of your putative
insert and label it by the random primer/32P method for at least 2.5
hours (light emmision systems will also work. You can also do this
while the filter is being processed.

1. place the filter on the plate containing your transformants until
the     filter wets.

2.  poke several holes in the filter edges with a needle (that has been
diped in india ink) all the way through and into the agar.  This is
essential to maintain the orientation of the filter when you go to pick

3.  lift the filter from the plate carefully (no smearing) and place it
colony side up on a paper towel.  Immediately return the plate to the
incubator so that the colonies recover (do not regrow the colonies
longer than 6 hours).

4.  place the filter in the autoclave and autoclave at a setting of
212F for ONE minute on SLOW exhaust.  The temp will exceed 212F but
don't worry about it this is normal.  This step lyses the cells, fixes
and denatures the DNA all in one step and is superior to chemical
methods for most purposes.

5.  remove the filter from the autoclave and wash it with 2XSSC for 10
minutes at 42C

6.  prehybe the filter as you do for a southern blot for one hour.

7.  hybridize your filter for 1-2 hours with your labeled probe

8.  quick-wash the filter with 2XSSC 0.1% SDS at RT and transfer into
the vessel that you normally use to wash blots.  Wash with 0.1XSSC,
0.1% SDS at 65C for 15 minutes

9.  Blot to dampness and place on film for one hour.

10. Match dots on film to colonies on the plate and pick.  

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