Creating Cell Banks

DATE

Staff Members

Printed Name Signature Initials

1.0 Preparation of Overnight Broth Culture Initial Check

1.1 Clean Biosafety cabinet Date:
Add Kanamycin Stock Solution Date:
(30mg/ml) to media at 1ml Kanamycin
Stock Solution per 1L of media
1.2 Aseptically transfer 50ml of media to a Date:
sterile flask
1.3 Store remaining media at 5±°C
1.4 Inoculate media in flask with a single colony from a streak plate using a sterile
loop.
1.5 Place flask in shaker and incubate for 16-20 hours at 37°C at 150 rpm.
1.6 Remove flask from shaker and ensure culture is turbid.

2.0 Preparation of Cell Bank Culture Initial Check

2.1 Clean Biosafety cabinet
2.2 Label two 2000ml sterile flasks with the cell bank number, date and initial
2.3 Aseptically transfer 500ml of media to each sterile flask
2.4 Aseptically transfer 10ml of overnight culture (Section 1.0)
Place flasks in shaker and incubate at
37°C
2.5 Incubate until OD600 is between 0.5 and 0.8
2.6 Measure the OD600 at regular intervals using a disposable cuvette and spectrophotometer.
Time OD600 Date Initial Check

3.0 Cell Bank Aliquoting and Storage Initial Check

3.1 Label 25 sterile vials
3.2 Transfer 50ml into each of 4 sterile conical tubes.
3.3 Centrifuge at 5000 rpm for 15 minutes at 5±° C.
3.4 Disgard supernatant and pool resuspended cell pellets in a total of 25ml of media
containing glycerol into a single conical tube.
3.5 Transfer 1.0 ml of bacterial suspension into labelled cryotubes and place into
cryostoarge containers.
3.6 Place cryostorage containers in -80°C freezer.

4.0 Cell Bank Analysis Initial Check

Clean Biosafety cabinet Date:
Place a frozen cell bank under the biosafety cabinet and allow it to thaw at room
temperature.
Streak the cell bank for isolation on: Plate 1 type:
agar plate without antibiotics and an
agar plate supplemented with Plate 2 type:
Kanamycin (30mg/ml).
Incubate plates at 37°C overnight Date:
Observe and record results below. Date:
Plate 1 type: Plate 2 type: