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Team:University of Chicago/Notebook/DH5-alpha competent cells
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(New page: ==Materials== ===TB Solution=== *10mM PIPES *15mM CaCl2 *250mM KCl *Dissolve in nanopure water and adjust pH 6.7 with KOH or HCL (solutes will dissolve as you do this) and then add 55mM M...)
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Revision as of 16:17, 7 August 2008
Contents |
Materials
TB Solution
- 10mM PIPES
- 15mM CaCl2
- 250mM KCl
- Dissolve in nanopure water and adjust pH 6.7 with KOH or HCL (solutes will dissolve as you do this) and then add
55mM MnCl2. Adjust to final volume. Sterilize by filtration with 0.45um filter and store at 4°C
LB Media
(per 1L batch)
- 30 g LB mix
- Dissolve in ddH2O, filling to 1L.
- for 20mM MgSO4 FIRST add 16mL 1M MgSO4 solution THEN fill to 1L
Procedure
1. Inoculate a 3ml overnight of E.coli in LB+20 mM MgSO4. 2. Next morning, inoculate 250 ml LB+20 mM Mg++ in a 2L flask with about 2ml overnight culture. Grow at room temp (23°C) with good aeration (250rpm) to an A600 of 0.4-0.6.
- Note: TEMPERATURE IS IMPORTANT! At 37C cells will grow up to proper OD in ~3 hours. A faster growing time, however, compromises efficiency, so choose temperature accordingly.
3. Place cells 10 min on ice. Transfer to a sterile bottle and spin 3K, 10', 4°C. 4. Resuspend pellet in 80 ml cold TB (swirl cells in bottle). Leave 10’/ice. 5. Spin cells 3K, 10', 4°C. 6. Resuspend cells in 20 ml cold TB then add 1.5 ml DMSO. Leave 10'/ice. 7. Dispense into 220 ul and 525 ul aliquots (in cold sterile tubes) and freeze in dry ice/EtOH bath. Store -70°C. Typically, competency about 5X 106 cfu/ug DNA. Note, improves after freezing. Cells good for a year and counting.
