Creating Competent E. coli Cells

Goal

• Goal: To create competent E. coli cells using either of the two methods listed below.

Pre-Protocol Questions

1. Do you know how to triple-streak cells onto a plate?
2. Do you have the necessary media plates (LB, Tryptic Soy, etc.)?
3. Do you know how to use the huge ultra-centrifuge in Superlab?

Current Version: CaCl2 Competent Cells without Sodium Ions (from Bash)

Special Reagents Needed

• TF1 Solution

1. 3mL 1M Potassium Acetate
2. 10mL 1M KCl
3. 2 mL 0.5M CaCl2
4. 5 mL 1M MnCl2
5. 18.75 mL 80% glycerol
6. Fill to 100 mL and adjust pH to 5.8 with 0.2M acetic acid or NaOH. Solution will change to slight brownish yellow color.
7. Filter sterilize only. Place in 3rd floor cold room.

• TF2 solution. Place in the 3rd floor cold room.

1. 1 mL 1M MOPS, pH 6.5
2. 1 mL 1M KCl
3. 15 mL 0.5M CaCl2
4. 18.75 mL 80% glycerol
5. Fill to 100mL with water and adjust pH to 6.5
6. Filter sterilize only. Place in 3rd floor cold room.

• Growth media

1. Fill a graduated cylinder to ~700ml with MilliQ water
2. Add 4g Yeast Extract in each flask
3. Add 16g Bactotryptone in each flask
4. Add 4g MgSO4 in each flask
5. Dissolve and fill up to 800ml with water
6. Adjust to pH 7.6 with KOH (not NaOH)
7. Put 400ml in each of two 2L flasks and immediately autoclave.

Day 1

1. Triple-streak previous competent cells for single colonies. Use a Tryptic Soy plate or an LB plate with antibiotics, if applicable.
2. Create TF1 solution
3. Create TF2 solution
4. Create Growth media
5. Create p1000 tips with 0.25cm cut off sterilely. Place in the 3rd floor cold room.
6. Sterilized large ultra-centrifuge tubes. Place in the 3rd floor cold room.
7. Place two 50ml Falcon tubes in the 3rd floor cold room.

Day 2

1. From this grown plate, pick a single colony and inoculate 10ml LB (+ antibiotics if applicable) in a 50ml Erlenmeyer flask. Grown overnight at 37 degrees with shaking.

Day 3

Important: Treat cells gently after addition of TF1 Solution — no vortexing or vigorous pipetting — as this will greatly reduce the competency.

1. Inoculate both 400ml LB flasks with 4.5ml of overnight growth and shake at 37 degrees.
2. Turn on the Superlab ultra-centrifuge, and chill to 4 degrees.
3. Turn on Superlab swinging rotor centrifuge, and chill to 4 degrees.
4. Measure OD after 1 hour and then every 30 minutes, until O.D. at 600nm is 0.2-0.4 (~3 hours). Using a 13mm tube, this translates to Ab 0.25-0.52.
5. Place flasks on ice, or even better, in an ice bath. Take up to 3rd floor cold room.
6. Put cells into four total large ultra-centrifuge tubes, and balance to 0.1g. This will be ~200ml each.
7. Pellet cells for 20 minutes at 3700 rpm / 3200 x g at 4 degrees in Superlab ultra-centrifuge.
8. Decant (gently pour off) supernatant and gently resuspend cells in 40ml ice cold TF1 solution per ultra-centrifuge tube by pipetting up and down with a serological pipette. Keep on ice.
9. Consolidate cells to two 50ml Falcon tubes and chill on ice for 20 minutes. Balance to 0.1g.
10. Pellet cells for 20 minutes at 3700 rpm at 4 degrees in the swinging rotor centrifuge.
11. Decant supernatant and gently resuspend cells in 2ml ice cold TF2 solution per pellet by pipetting up and down with a cut-off p1000 pipette tip. Chill on ice for 10 minutes. Consolidate into one tube.
12. In cold room, aliquot 50 ul into the 0.6ml tubes, using the cut-off p1000 tips and the electronic pipettor.
13. Flash freeze aliquots in liquid nitrogen.

Old Version: Traditional CaCl2 Competent Cells

Special Reagents Needed

50ml of 0.1M CaCl2 in 15% glycerol (40.7ml 0.1M CaCl2 + 9.3ml of 80% glycerol), autoclaved (for Day 3)

Day 1

1. Triple-streak previous competent cells for single colonies. Use a Tryptic Soy plate or an LB plate with antibiotics, if applicable.

Day 2

1. From this grown plate, pick a single colony and inoculate 10ml LB (+ antibiotics if applicable) in a 50ml Erlenmeyer flask. Grown overnight at 37 degrees with shaking.

a. 37 degree shaking incubator located next to the two fridges.

1. Have a 2L Erlenmeyer flask with 500mL LB autoclaved and ready to go for tomorrow.
2. Have three 250ml centrifuge tubes autoclaved and ready to go. Ask Eric where these are.
3. Have 50ml of 0.1M CaCl2 in 15% glycerol sterilised and ready to go.
4. Autoclave
5. Label and set up 100 0.6ml tubes for the eventual aliquots.

Day 3

Important: Treat cells gently after addition of CaCl2 — no vortexing or vigorous pipetting — as this will greatly reduce the competency.

1. Transfer 5ml of this overnight culture into the 2L flask containing 500ml LB. Grow at 37 degrees with shaking.
2. Immediately after transferring this culture, sterile syringe filter (0.22um filter) the 50ml 0.1M CaCl2 in glycerol. Put on ice and place at 4 degrees. Additionally, chill two 50ml plastic tubes.
3. Grow culture until OD600 is ~0.4. (Use spectrophotometer to check the OD.) This will take 2-3 hours.
4. From now on, everything must be on ice!
5. Roughly divide the 500ml of grown E. coli into the three 250ml centrifuge tubes. Do not be tempted to only use 2 tubes; this will result in LB spilling out into the centrifuge.
6. Put these tubes in a styrofoam box containing ice; place in the refrigerator. These need to incubate for a total of 20 minutes.
7. Go to Superlab and turn on the large centrifuge. Ensure that it is set to 4 degrees and that it is actively decreasing in temperature.
8. Also, turn on the tabletop swinging bucket centrifuge in Superlab. Ensure that it is set to 4 degrees and that it is actively decreasing in temperature.
9. After the 20-minute incubation at 4 degrees, weigh out the three centrifuge tubes and balance them to the nearest 0.5g. This will take some time; keep the E. coli on ice.
10. Centrifuge the cultures at 3000 x g at 4 degrees for 10 minutes.
11. Turn off centrifuge. Take cultures back to the MEE lab and slowly pour off supernatant into a large graduated beaker.
12. Resuspend the cells gently in 30ml (total) ice-cold 0.1M CaCl2 + glycerol. Transfer to the two pre-chilled 50ml tubes.
13. Incubate cells on ice for 30 minutes at 4 degrees.
14. After the 30-minute incubation at 4 degrees, weigh out the two 50ml tubes and balance them to the nearest 0.5g.
15. Centrifuge for 5 minutes at 3000 x g at 4 degrees using the swinging bucket centrifuge in Superlab.
16. Turn off centrifuge. Take cultures back to the MEE lab and slowly pour off supernatant into same large graduated beaker as before. When you have time, add enough Virkon to this to kill off any cells and dispose of.
17. Resuspend cells gently with 8ml total ice-cold 0.1M CaCl2 in glycerol. Do not vortex!
18. Transfer 260ul aliquots into 0.6ml tubes; flash-freeze in liquid nitrogen and store at -80 degrees. Use the electronic pipettor to do this faster!