Strange PCR colony screen result

Eric C. Anderson anderson at pharmdec.wustl.edu
Thu Mar 16 13:43:48 EST 1995


here's a result i haven't come across yet in 2 years of doing this stuff. 
i just screened a bunch of E. coli transformants with T7 and M13 primers
and got the following results:

1.  i have a number of good, positive clones that all match my positive
control.  single, strong band at about 1250bp.  (this is the good part.)
2.  my negative colony controls (blue colonies on blue/white selection) all
give the right size band and it is the same as the vector alone colonies. 
(this is also good.)
3.  the colonies that don't look good have a number of different multiple
banding patterns with nothing the correct size.
4.  my no dna, water control has a banding pattern that is unlike anything
else on the gel.  it should of course be blank, but since it isn't, i
expected it to look like one of my incorrect transformants or like my
positive or negative controls.  but it's got a patter all it's own that
i've never seen before with this set of primers.  if there's contamination,
i can't figure out why i didn't get that pattern in any of my other lanes
along with the correct sized band?

just by guesstimating, i would say that the correct transformants and
positive controls have about 5-10X as much product as this water control,
but that it has about 2X as much product as my negative controls.

any suggestions about what's going on here would be appreciated.  as per a
number of recent posts, i picked the colonies with pipet tips instead of
toothpicks and got much cleaner and more helpful results (the above problem
notwithstanding).

thanks in advance,

eric 

-- 
eric c. anderson                                
anderson at pharmdec.wustl.edu
dept. of molecular bio. and pharm.               (314)362-3963 (lab)
washington univ. school of medicine              (314)862-2435 (home)
660 s. euclid box 8103                           (314)362-7058 (FAX)
st. louis, mo 63110



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