1.
On
the bottom of each petri dish, label 8 colonies to be tested. The
colonies
should be white.
Blue colonies
are producing the enzyme Beta-galactosidase,
which breaks
down X-gal in the media into a blue compound. These
colonies are able to produce
this enzyme while white colonies are not. Beta-galactosidase
synthesis is
interrupted in white colonies because we have inserted DNA right into
the
middle of its gene, lacZ.
This is called blue/white screening, and means that white colonies
should
have an insert, while blue colonies do not.
2.
Label
the bottom of a fresh petri dish with 8 squares to transfer colonies
into.
3.
For
each colony, put 15 µl of water in 0.2
mL strip tubes. Label
them with the colony number. Prepare 5 ml cultures of LB broth for each
colony
and add 10 µl of Ampicillin
(25 mg/mL).
4.
Take
a 20 µl
pipet tip and touch a colony very lightly and dip the tip a
couple of times into
the 15 µl
of water, then with the same tip, streak onto the
fresh media in the
appropriate square and put into LB broth. Repeat for each colony.
5.
Heat
the strip tubes in the PCR machine for 5 minutes at 95°C. Place
tubes on ice
immediately afterwards.
6.
Thaw
PCR reagents. Fill out PCR sheet, with 10 µl total volume and
1 µl of colony DNA for
each
reaction. Use primers M13F and M13R, these bind to the plasmid DNA on
either
side of the insert and amplify across it. This will help to determine
what size
the insert is.
7.
Make
a master mix according to the calculations above, with all reagents
except for
the DNA. Dispense the master mix into strip tubes (9 µl each) and then
add the DNA
prepared in step 4 (1 µl
each). Label tubes and put in PCR
machine.
Materials
Necessary
Plates
from cloning (Invitrogen/Promega)
8-strip
or single 0.2 µl PCR tubes
Sterilized
DI water
10
and 20 µl
pipette tips
Equipment
Necessary
10 µl
micropipette
20 µl
micropipette
PCR
Thermocycler