reliable colony screening w/PCR?
dkim at nmsu.edu
Mon Jun 2 06:29:25 EST 1997
There are many PCR colony screening protocols to be followed, but I find
that most require elaborate steps for liberating plasmids from the
bacteria. Often this involves a suspension/boiling step before loading
the template into the reactions.
I wonder why this is done, as the PCR reaction repeatedly brings the
sample to near-boiling temperatures with every cycle.
When screening colonies, what I do is:
1. Prepare enough PCR reaction mix for all the colonies I intend to
screen, assuming a 10 microliter reaction volume per colony (10 colonies
requires 100 microliters of reaction mix). Divide this into reaction
tubes (do not add oil)
2. Prepare a bacterial culture plate with a grid for placing picked
3. Take the primary transformation plate and touch a pipet tip to a
colony. Touch the gridded plate, then place the tip into the first tube.
4. Repeat for each colony.
5. Remove the pipet tips and discard. Add oil, if necessary, then
cap the tubes and run the PCR cycles.
6. When complete, add gel loading dye and load the reactions onto a
gel. Usually only a few microliters is necessary.
I find this works for me almost every time. I am using only a little
enzyme because of the small reaction volumes. I have successfully
screened colonies in as little as 5 microliters of PCR reaction volume
each, loading the entire sample on the gel. By the time the reactions are
done, there is visible bacteria in the gridded plate, so I can proceed
with overnight cultures, or whatever I have planned next.
Do not be bound by "lab traditions" to do your work the same way always.
Take a few chances! It is not difficult to imagine a PCR colony screening
procedure. Such a simple thing can be tested in the course of an
afternoon, and is good practice for when you are developing the next big
dkim at nmsu.edu
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