Overnight
cultures and archiving of clones
1. Prepare an archive LB + ampicillin +
X-gal + IPTG plate by
making a grid on the back of the Petri dish with a felt pen. Grid cells
should
be approximately 1 x 1 cm; number each cell.
2. For each colony you intend to test,
fill a 0.2 mL PCR
tube with 50 µl sterile water. Number the tubes.
3. Select single white colonies from
plates with a
pipette tip. Gently streak the colony across a grid on the other plate.
Do not
discard the tip.
4. Take the same tip and place it in the
appropriately numbered PCR tube.
5. Repeat for each colony you intend to
test.
6. Cover the plate and incubate at 37° C overnight. Remove the pipette tips from
the tubes.
7. Place the tubes in a thermocycler.
Incubate at 99° C for 10 minutes.
8. Cool the tubes to room temperature and
proceed
with colony PCR.
Growing
cells for
Plasmid Isolation
1. For each colony to be sequenced,
prepare a 15 mL
tube with 5 mL of LB broth with 100 µg/mL of ampicillin.
2. Select colonies with known inserts and
touch with
a pipette tip.
3. Place the tip in LB culture and grow at
37° C shaking incubator overnight. Use this
culture in
the plasmid isolation protocols (1,2).
Materials
necessary
15
ml culture tubes
100
mg/ml ampicillin stock
LB
+ amp + X-gal + IPTG plates
0.2
ml PCR tubes
Sterile
DI water
10,
20, and 1000 µl pipette tips
Equipment
necessary
37° C
incubator
37° C
shaking incubator
1000 µL micropipette
10 µl micropipette