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From 2012.igem.org

Contents 1 Preparation of electrocompetent cells of E.coli 1.1 Reagents and Materials 1.2 Procedure 1.2.1 Day 1: 1.2.2 Day 2: 1.2.3 Day 3:

Preparation of electrocompetent cells of E.coli

Reagents and Materials

For 25 samples:

Reagents:

• 1L SOB medium
• 400 ml MQ water
• 250ml 10% glycerol

Materials:

• Yellow and blue pipette tips and the yellow blue pipettes
• Eppendorf tubes
• Greiner tubes
• disposable 10 ml pipettes
• an automatic pipette
• the SLA 3000 rotor
• scales
• coldroom
• -80 freezer
• -80 boxes

Procedure

Day 1:

• Inoculate appropriate strain from -80 degree Celcius stock to a LB agar plate and incubate at 37 degree Celcius o/n.

Day 2:

• Inoculate 10 ml SOB medium with a single colony from the plate and incubate o/n shaking at 37 degree Celcius.

Day 3:

preparations in advance (can be made while cells are growing):

1. Take two or three big buckets of ice and add a little water. Leave them at RT for a while so that a layer of ice-cold water will form at the bottom.
2. Put 50 empty eppendorf tubes (closed caps) in a box and store them at -80 degree Celcius.
3. Place your glycerol, MQ and empty Beckmann-tubes on melting ice in the cold room. Make sure you have bucket of melting ice ready to chill your cells in. Furthermore, make sure you have in the cold room:
   • Yellow and blue pipette tips and the yellow blue pipettes
   • one disposable 10 ml pipette
   • an automatic pipette
   • the SLA 3000 rotor
   • scales

Half an hour before your cells are at their desired OD (approximately 2.5 hours after inoculation), switch on the Beckmann-centrifuge and pre-chill it at 4 degree Celcius.

1. Take 1 ml of SOB out of the Erlenmeyer and put it in a 1 ml cuvette (for blanking the SmartSpec).
2. Inoculate 2*500 ml SOB in 2 L Erlenmeyer flasks with each 1 ml preculture.
3. Incubate ~3-5 h shaking at 130 RPM at 37 degree Celcius.
4. Shake until A600 is 0.55-0.6, this will take about 4 hours (Never over 0.65! - better to be little under 0.6), Check A600 of culture by removing a 1 ml sample and reading on SmartSpec.
5. Chill the Erlenmeyer on ice for about 30-45 minutes (check when A600 is 0.5-0.6), then quickly! spin down in sterile 250ml bottles in SLA3000 rotor in Bechmann centrifuge at 3500 RPM and 4 degree Celcius for 10 minutes. From this point on keep the cells on ice at all times when carrying between the centrifuge and the cold room. Place on ice instantly upon removal from centrifuge. Do all work in the cold room
6. Decant supernatant and hold upside down for a few seconds to allow all supernatant to drain off. Immediatly put the bottle into ice. Resuspend the pellets by adding 10-20 ml of ICE cold sterile water and swirling (don't use your pipette too much as this may affect the transformation efficiency! until entire pellet is resuspended. Add ICE cold sterile water to the resuspension to a total volume of 100 ml and combine the suspensions in 2 buckets, recentrifuge at the same parameters as above.
7. Repeat former step wash using 10% glycerol,combine in 1 tube.
8. Decant and resuspend pellet in 10 ml (use a 25 ml pipette) 10% ice cold glycerol by swirling and transfer into Greiner tube. Centrifuge again at 3500 RPM and 4 degree Celcius for 5 minutes. While rotor is slowing down bring rack of -80 degree Celcius microfuge tubes into the cold room.
9. Decant (carefully!) and resuspend pellet in 1.0 ml of ice cold 10/20% glycerol by swirling until entire pellet is resuspended. Another option is to resuspend the cells in the glycerol remaining at the bottom of the Greiner tube using a 1000uL pipette and add extra glycerol solution to a final volume of 1 ml.
10. Optional: for better results you can make a final OD measurement. Make a 1:1000 dilution in LB or SOB. A600 should be around 0.10-0.15. If spin the cells down and remove some of the supernatant, resuspend and measure again. If they are too concentrated, add 10% glycerol to reach the desired OD. This could mean that you for example end up with 2 ml cells from your culture.
11. In the cold room: Distribute in aliquots of 40 ul to Eppendorf tubes. (use repeater pipette to save time and prevent RSI.) if your microfuge tubes aren't frozen anymore, flash freeze the aliquots using liquid nitrogen.
12. Label a container with the name of the strain, user and date and store at -80 degree Celcius.