Colony screening

Tim Fitzwater tfitzwater at
Mon Jul 2 09:13:25 EST 2001

Typical ligation protocols utilize 5 pmol of insert PCR product in a 20 µL
reaction (0.25 pmol/µL) with 0.5 pmols of vector.  Assuming that all the
vectors acquire a single copy of insert, 4.5 pmols (0.225 pmol/µL) of excess
insert remain in the ligation reaction.  2 µL of a 1:5 dilution of the
ligation reaction (0.09 pmol) is transformed and diluted 1:9 with SOC media
(0.01 pmol/µL).  If 100 µL (1.0 pmol) is spread on a 55.4 cm2 plate (0.018
pmol/mm2) and a 4 mm2 agar pick is used, then 0.072 pmols (4.35 x 1010
molecules) of background insert are available for the colony PCR assay.
Using at least one primer that hybridizes to the vector avoids amplification
of this contaminating insert.  

Tim Fitzwater
SomaLogic, Inc.
Boulder, CO


More information about the Methods mailing list