Gene Cloning

134 Gene Cloning
a
b
c
d
e
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h
1 2 3 4 5 6 7 8 9 10 11 12
Figure 5.8 PCR screening of pools from a library. Pools are made by
taking a small sample from each well in, for instance, row a and mixing them; this
is pool a. The process is repeated for rows b–h and for columns 1–12 giving a
total of 20 pools. DNA extracted from each pool is used as the template in a
PCR reaction and the reactions are analyzed by agarose gel electrophoresis. If a
band is seen for DNA extracted from pools 4 and c, a single well in the multiwell
plate can be identified containing the clone that gave rise to the band.
understand if we start by considering a small library consisting of 96 clones,
each of which has been inoculated into a separate well in a multi-well plate
(Figure 5.8). A positive signal in the pool created by mixing all of the clones
in row c and in the pool created from all of the clones in column 4 gives a
unique address to the well where row c and column 4 intersect. In this way
any individual clone can be identified from 20 PCR reactions rather than
performing one for each of the 96 wells. This principle can be extended so
that each clone has an address in three or even four dimensions rather than
only in two, allowing 9600 clones to be screened in only 40 PCR reactions.
To prepare a library for screening by this method it needs to be gridded
out in multi-well plates. This can be done in one of two ways. The library
can be plated as plaques or colonies on agar plates and individually inoculated
into the wells of the multi-well plate. This is a labor intensive process
and, because it involves the experimenter inoculating wells with individual
clones, it can lead to bias in favor of larger colonies or plaques. The alternative
method involves diluting the library. A small part of the original
library, the packaging mix in the case of a phage library, or transformation
in the case of a plasmid library, is plated out and the titer of the library calculated
(see Section 4.13). A larger sample is then diluted to give a titer of
100 colonies per mL. Dispensing 100 μL into each well will theoretically
give a 10 clones in each well. These are then pooled as described above and
PCR reactions performed to identify the wells containing the clone of interest.
This technique is often used to screen commercially available libraries
which can be supplied ready gridded.