Subcloning in 96-well Plates - Sibley Lab

Subcloning in 96-well Plates
Sibley Lab
11/24/03
JS
1) Plate HFF cells in an appropriate number of 96-well plates and grow to
confluence.
2) Harvest a freshly lysed T25 of Toxo cells.
3) Resuspend the pellet in 10 ml of D10 and count the cell concentration on
a hemocytometer.
4) After calculating the concentration, dilute the cell suspension so that there
is one cell per 100µl of media.
5) Remove the old media from the 96-well plates and add 100µl of fresh D10
to each well.
6) Then add 100µl of the diluted cell suspension to each well.
7) Spin the plates in a microplate carrier for 5 min. at 1200rpm using a tabletop
centrifuge (this will force the toxo cells to the bottom so that they can
plate themselves more easily).
8) Allow the cells to grow in the CO 2 incubator for 5-7 days. DO NOT MOVE
THE PLATES while they are incubating.
9) Score the wells by examining the plate under the inverted microscope
using the 10-20X phase lens. Mark the wells containing single plaques as
defined by a clear area of host cell lysis surrounded by heavily infected
cells.
10) Dump the media and feed the positives with D10. Mixing helps the
plaques to spread.
11) After 2-5 days the entire well should be lysed out. Transfer 10% of the
contents to new 96-well plates containing confluent HFF cells. (From
each positive well you should transfer 10% to a 96-well plate for freezing
and another 10% to a separate 96-well plate that will be used for PCR.)
12) Spin the plates as done previously in the table-top centrifuge and place in
CO 2 incubator.
13) After 1-2 days of incubation prepare the designated plates for freezing.
Add DMSO to a concentration of 10% and seal the plate with adhesive
plate sealer. Place in the -80°C freezer for storage.

Sibley Lab
11/24/03
JS
14) After another 4-5 days of incubation in the CO 2 incubator, the remaining
plates should have lysed the monolayer completely.
15) Prepare PCR lysate from each well and perform PCR using appropriate
primers. Be sure to include control.
16) Run out PCR samples and analyze.
Flow Chart
T25 – freshly lysed toxo
Harvest/Count/Dilute
One cell per well
Incubate one week
Mark positives
and feed
Incubate 2-5 days
Transfer 10% Transfer 10%
from each + well
from each + well
Incubate 2-3 days and
freeze with 10% DMSO
Incubate 5-7 days
until lysis, then PCR
Recover and expand chosen wells