Competent e coli

TFB Protocol for DH5a

1. Inoculate a single colony from an LB plate into 5ml of LB medium in a plating tube OR inoculate about 1 µL of competent E coli into 5 mL LB broth. Incubate overnight at 37°C with shaking (approximately 225rpm).
   
2. Subculture the overnight culture 1:100 by inoculating 5 mL into 250 mL of LB supplemented with 20mM MgSO₄ (0.6 g). Grow the cells in a 1 L flask until the OD600 reaches 0.4-0.6 (usually 5-6 hours, but the time may vary).
3. Pellet the cells by centrifugation at 4,500 x g for 5 minutes at 4°C. For a 250 mL culture, use two 250ml centrifuge bottles in a large rotor.
   
4. Gently resuspend the cell pellet in 0.4 original volume of ice-cold TFB1. For a 250ml subculture, use 100ml of TFB1 (50 mL/bottle). Combine the resuspended cells in one bottle. For the remaining steps, keep the cells on ice and chill all pipettes, tubes and flasks.
   
5. Incubate the resuspended cells on ice for 15 minutes.
   
6. Pellet the cells by centrifugation at 4,500 x g for 5 minutes at 4°C.
   
7. Gently resuspend the cells in 1/25 original volume of ice-cold TFB2. For a 250ml subculture, use 10ml of TFB2.
   
8. Incubate the cells on ice for 15-60 minutes, then aliquot 100 µL/tube for storage at -80°C. Quick-freeze the tubes. JM109 competent cells prepared by this method are stable for 1 year.
   

Note: Competent cells may be conveniently frozen in aliquot of 100 µL cells per tube. I typically transform 50 µL cells with 2-10 µL of a ligation reaction, and you should get between 1x10⁸ to 1x10⁹ cfu's/ug DNA.

Recipes

LB broth (PSI broth)

For 1 liter:

• 5g Bacto yeast extract
• 20g Bacto Tryptone
• 5g Sodium Chloride (NaCl)

pH 7.6 with potassium hydroxide

TFB1

For 200ml:

• 0.588g potassium acetate
• 2.42g rubidium chloride
• 0.294g calcium chloride
• 2.0g manganese chloride
• 30ml glycerol

pH 5.8 with dilute acetic acid

TFB2

For 100ml:

• 0.21g MOPS
• 1.1g calcium chloride
• 0.121g rubidium chloride
• 15ml glycerol

pH 6.5 with dilute NaOH

CaCl₂ Protocol for BL21 & ZYCY10P3S2T

Prepare two simple solutions:

Solution 1

• 0.1 M CaCl₂ (1.11g in 100 mL)

Solution 2

• 0.1 M CaCl₂ + 15% glycerol (1.11g in 85 mL ddH₂O + 15 mL glycerol)

Solutions may be autoclaved, but filter-sterilization has been shown to yield better results

Protocol

The night before:

• Grow a culture of bacteria in 1-5 mL SOB (or LB) overnight

Ensure all solutions are ice cold

• Add 1-2 mL of overnight culture to 100 mL of fresh LB medium and record starting OD₆₀₀
• Grow for 2-4 hours at 37^oC with shaking, until culture OD₆₀₀ is 0.4-0.6
• Pellet the culture by centrifuging at >4000g for 5-10 minutes at 4^oC
• Re-suspend the pellet in 20 mL Solution 1 and incubate for 30 minutes on ice with gentle shaking
• Pellet the culture by centrifuging at >4000g for 5-10 minutes at 4^oC
• Re-suspend the pellet in 5 mL Solution 2 and snap-freeze 100 uL aliquots in LN₂ before storing in -80^oC freezer