Day 1: a) Fill each well of a
96-well plate SM/2 broth with chloramphenicol, 20ug/ml.
b) Add 10ul cosmid clone from a 96-well plate to each well
Get the plate from the -80 freezer. Allow enough time for the plate
contents to thaw and inoculate the new plate with 10ul from each
well using a multichannel pipettor. Keep the inoculum on the new plate in
exactly the same orientation as the plate from the freezer. Put a check
on the original plate and replace it in the -80 freezer.
c)
Incubate the subculture plate at 37 overnight.
Day 2: a) Scrape
the Dictyostelium fruiting bodies from a bacterial plate and suspend in
1ml sterile water. We use E. coli B/r.
b) Count the Dicty fruiting bodies
using a hemocytometer, and dilute to 5 Dicty cells/ul.
c) Add
40ul of the above Dicty diln to each well of the subculture plate, giving a
total of 200 Dicty cells/well.
Each well of the 96-well subculture plate will thus have 10ul of overnight
enriched cosmid clone and 200 Dicty cells.
d) Using an 8-channel multichannel pipettor, inoculate SM/2, Cm20 agar
plates with 10ul cosmid clone/Dicty mixture.
Each 90mm plate can hold 4 rows of inoculum, A1-H1, A2-H2, etc.
e) Allow the inoculum spots to dry on the plate before removing the
plates to incubate at room temp in the dark.
f) Spread each of 4 plates with 30ul from the A1 control well on the
96-well plate and incubate at RT in the dark.
g)
Incubate 4-6 days.
No comments:
Post a Comment