Electrocompetent CB2A Cell Preparation

Objective

Author(s): Jessie Luo, Ada Jiang

Preparation of CB2A cells to create electrocompetent cells, which are receptive to DNA uptake via electroporation for construct expression. This protocol is adapted from a protocol provided by our advisor, Beth.

Materials

Colony plates

CB2A PYE plate colonies

Media/Solutions

(550 mL) PYE liquid medium
(1 L) Distilled or Milli-Q water, sterile
(55 mL) 10% glycerol

Equipment

250 mL sterile flask with foil
2 L sterile flask with foil
10 sterile Falcon tubes
50 sterile 1.5 mL Eppendorf tubes
1 cm cuvettes for spectrometer
Pipette boy and 10 mL sterile pipettes
Low-speed centrifuge

Procedure

Set-up

Day 0 - Pre-culture

Inoculate 50 mL of PYE culture in a 250 mL flask with a CB2A colony.
Shake at 30˚C for ~48 hours. If incubating for over 48 hours, you can grow the culture on the bench at room temperature until the appropriate optical density at 600 nm (OD600) is reached.

Day 2 - Culture set up

Early morning: Inoculate culture with pre-culture.
1. Use a spectrometer to find the pre-culture OD600.
2. Fill a 2 L flask with 500 mL of PYE media.
3. Add the correct volume of pre-culture to make OD600 0.1 in 500 mL of PYE. If pre-culture OD5, add 10 mL pre-culture to 480 mL fresh PYE culture for OD0.1 (1:50 dilution)
Shake at 30˚C until OD600 is 0.3-0.6. This should take 3-7 hours.

Electrocompetent cell preparation

Wash Steps

Pour culture into 50 mL Falcon tubes (10), in the flow hood or sterile environment.
Centrifuge the culture at 3700 xg for 20 min.
Pipette out the culture supernatant and discard (pouring may also work but may dislodge the pellet - be careful if doing this).
Add about 5 mL sterile cold water to each bottle and resuspend with a 10 mL pipette carefully.
Then, fill the bottles to the top with sterile cold water (“full volume wash”) and centrifuge at 3700 xg for 20 minutes.
Pipette off supernatant (note: pellets tend to be looser after the water washes).
Repeat the process for suspension of each pellet in 2 mL sterile cold water
Fill bottles approximately half full with sterile cold water (“half wash”).
Centrifuge at 3700 xg for 20 min
Pipette out and discard supernatant.

Glycerol Steps

Suspend pellets (10) in 5 mL 10% glycerol each.
Combine suspensions into 2 Falcon tubes and centrifuge at 3700 xg for 15 min.
Discard supernatant.
Resuspend pellets (2) in 0.75 mL 10% glycerol each.
Aliquot 50 μL portions into sterile 1.7 ml Eppendorf tubes and freeze in -70 to 80°C.