plasmid screening

Matthew L. Brown, Ph.D gemini3 at mindspring.com
Thu Mar 20 19:54:20 EST 1997


DzungDiep,
	I currently use two different methods for screening recombinant
plasmids. One is good if you're in a hurry and have lots of money. The
other is good if you can wait a day and don't have much money. I don't
know which of these statements applies to me. Probably the latter.
Anyway...

Protocol 1: Colony PCR. Pick individual colonies and scratch onto
replica plate with a sterile eppendorf pipet tip. Resuspend remaining
material from colony in ~50 uL sterile water. Boil 5 min. Centrifuge at
high speed and remove a 10 uL aliquot for PCR. Add primers, dNTPs, Taq,
and buffer then cycle as you normally might for PCR. As far as the
primers go, you can use some type of vector arm primers like M13 forward
and reverse or use genespecific sequences if you know them or if the
clone was derived by PCR use the primers that generated the original PCR
product. Analyze products on an agarose gel.
Protocol 2: Cracking Assay (similar to, but distinct from that in Blue
Books). Pick individual colonies and scratch onto replica plate with a
sterile eppendorf pipet tip. Always include a clone that is known not to
contain an insert (i.e. vector alone). Incubate overnight. Resuspend a
good loopful of scratched colony in ~25 uL of 10mM EDTA. Add ~25 uL of a
fresh solution of 0.2 N NaOH, 0.5% SDS, 20% sucrose. Mix then heat at 70
C for 5 min. Cool to room temp slowly then add ~5 uL of 4 M KCl. Mix and
incubate on ice 5 min. Centrifuge for 3 min at high speed (i.e.
12,000xG). Load ~ 25 uL of each supernatant in wells of an 8-well 1%
agarose gel and electrophorese. Always include on each gel a "cracked"
sample containing vector alone and a ladder of course. In Maniatis,
Fritsch, and Sambrook's book there is a protocol similar to this that
suggests that a 1 mm colony can be replica plated and cracked directly
from the remaining colony material lifted. I have found this not to be
the case. In my hands this procedure only works when the amount of
starting material (i.e. bacterial colony) is more substantial (like a
good size loopful).
Hope these help. Let me know.

Matthew L. Brown, Ph.D.
Univ. of Alabama at Birmingham
Dept. of Medicine



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