Protocol: Preparation for Ultra competent cells of E.Coli DH5-alpha

Preparation:

• Prepare LB media and Transformation Buffer.
  

  Transformation Buffer

  10 mM Hepes
  15 mM CaCl₂
  250 mM KCl (pH to 6.7 with KOH)
  MIX Thoroughly with constant stirring and than add 55 mM MnCl2 and filter sterilize through a 0.2 micron filter, store at 4°C

• Sterilize two 300 mL centrifuge bottles; 100 mL graduated cylinder.
• Pre-cool the rotor, buffer, and sterile plastic ware as well as sterile 25 mL pipet, micropipet tips and microcentrifuge tubes in cold cabinet or deep refrigerator.

Day 1:

1. Take 25 μL of glycerol stock and inoculate a 10 mL LB  broth. Grow overnight at 37°C. 

Day 2:

1. Inoculate 500 mL flasks containing 200 mL LB broth with 1 mL of the overnight culture. Grow at 22°C, shaking at 200-250 rpm until

OD₆₀₀ = 0.5 (This may take up to 3 days.)

Final Day:

First Spin -

1. Transfer contents of each flask to a centrifuge bottle and balance them.
2. Incubate bottles on ice for 20 minutes.
3. Spin at 4100 rpm (2500 X G) and 4°C for 10 minutes.
4. Place bottles on ice and take to Laminar Air Flow/Bio Safety Cabinet.

First Resuspension – On Ice (Should be done very gently and constantly keeping it on ice in between, making sure that it remains cold)

1. Pour off supernatant into flasks and invert bottles on paper towels.
2. Gently resuspend pellets (by swirling) in Transformation Buffer (1/3 original volume).
3. Incubate on ice 15 minutes. 

Second Spin

1. Spin down cells exactly as before.
2. Place bottles on ice and take to Laminar Air Flow/Bio Safety Cabinet.

Final Resuspension – ON ICE

1. Pour off supernatant into flasks as before.
2. Resuspend pellets in Transformation Buffer (1/12 original volume).
3. Add DMSO (5% final concentration), swirling gently.
4. Incubate on ice 10 minutes.
5. Dispense 110 μL cells into pre-cooled microcentrifuge tubes.
6. Freeze with liquid N, and store immediately in -80°C. 

NOTE:

1. Don’t try to pre-cool the DMSO. It will get frozen