IGEM:Brown/2007/Lab Protocols/Ultra Competent E coli prep Brodsky Updated

Procedure

1. Inoculate a 10 mL of LB with a single E coli colony for an overnight culture. (its okay to inoculate directly from a frozen stock as well.)
2. Dilute 10 mL into 250 mL SOB medium and grow at 25*C to OD600 = .6. (This will take 5-8 hours for DH5alpha and XL1-Blue). This should be done in a baffle flask if available, and otherwise in a large flask that can provide sufficient aeration but cannot be contaminated.
3. Transfer culture to clean sterile 250 mL bottle and place on ice for 10 mins. (the bottles should be rinsed with ddH2O and autoclaved while filled with ddH2O to get rid of all traces of detergent.)
4. Spin at 2500g for 10 mins at 4*C.
5. Decant supernatant and using a sterile plastic 25mL pipette, gently resuspend cells in 80mL of ice-cold TB. Do this by first adding 10 mL TB, and slowly pipetting up and down, and once they are mixed you can add the remaining 70 mL TB and mix by swirling the bottle. Keep in mind the cells are very fragile at this point.
6. place on ice for 10 mins.
7. Spin at 2500g for 10 mins at 4*C.
8. Decant supernatant and using a sterile plastic 25mL pipette, gently resuspend cells in 20mL of ice-cold TB + 1.5 mL DMSO. (DMSO does not need to be sterilized)
9. place on ice for 10 mins.
10. aliquot 50-100uL per tube and immediately freeze in liquid nitrogen.
11. Store aliquots at -80*C.

Notes: Reagents:

SOB:

1. 2% Trytone
2. 0.5% Yeast Extract
3. 10mM NaCl
4. 2.5 mM KCl
5. 10 mM MgCl2 * 4 H2O
6. 10 mM MgSO4 * 7 H2O
7. Filter sterilize: Do not Autoclave!

for 1 liter use:

1. 20g Trytone
2. 5g Yeast Extract
3. 0.584g NaCl
4. 0.186g KCl
5. 2.033g MgCl2 * 4 H2O
6. 2.468g MgSO4 * 7 H2O
7. Filter sterilize: Do not Autoclave!

TB:

1. 10mM Pipes pH 7.0
2. 55mM of MnCl2*4 H2O
3. 15 mM of CaCl2* 2 H2O
4. 250 mM of KCl
5. Filter Sterilize at 4*C.

for 500mL use:

1. 5mL of 1 M stock Pipes pH 7.0
2. 5.44g of MnCl2*4 H2O
3. 7.5mL of 1M stock of CaCl2* 2 H2O
4. 9.32g of KCl
5. Filter Sterilize at 4*C.