Griffitts:Chemically competent cells: Difference between revisions
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===Mg<sup>2+</sup>/Ca<sup>2+</sup>=== | ===Mg<sup>2+</sup>/Ca<sup>2+</sup>=== | ||
* 3.25 g MgCl<sub>2</sub>·6H<sub>2</sub>O | * 3.25 g MgCl<sub>2</sub>·6H<sub>2</sub>O | ||
* 0.6 g CaCl<sub>2</sub>·2H<sub>2</sub>O | * 0.6 g CaCl<sub>2</sub>·2H<sub>2</sub>O | ||
* 200 mL dH<sub>2</sub>O<br> | * 200 mL dH<sub>2</sub>O<br> | ||
Autoclave | Autoclave | ||
===100 mM Calcium Chloride=== | ===100 mM Calcium Chloride=== | ||
* 2.95 g CaCl<sub>2</sub>·2H<sub>2</sub>O | * 2.95 g CaCl<sub>2</sub>·2H<sub>2</sub>O | ||
* 200 mL dH<sub>2</sub>O<br> | * 200 mL dH<sub>2</sub>O<br> | ||
Autoclave | Autoclave |
Revision as of 14:40, 28 January 2008
Procedure
Note: Cells should never touch anything that is warm—chill solutions, pipets, tubes, etc. beforehand
- Grow DH5α overnight in 5 mL LB at 37°C
- Inoculate 100 mL LB with 1 mL of saturated overnight culture of DH5α
- Shake at 37°C until OD600 = 0.4
- Usually 2-3 hours
- Place in an ice bath for 5 minutes
- Divide the culture into 2 tubes with ~40 mL each
- Centrifuge at 8000 rpm for 10 minutes in a cold SS34 rotor
- The Sorvall Centrifuge is found in the Harker/Breakwell lab (747 WIDB)
- Gently resuspend each pellet with 15 mL of cold Mg2+/Ca2+ solution
- Incubate in an ice bath for 30 minutes
- Centrifuge at 8000 rpm for 10 minutes
- Resuspend each pellet with 1.6 mL of cold 100mM CaCl2 solution
- Incubate in an ice bath for 20 minutes
- Combine cells to one tube
- Add 0.5 mL cold 80% glycerol and swirl to mix
- Flash-freeze in liquid nitrogen as 100-μL aliquots
- Store in the -80°C freezer
Transformation
- Add 1 μL plasmid to chemocompetent DH5α cells
- Incubate on ice for 30 minutes
- Heat shock at 42°C for 1-2 minutes
- Add 800 μL warm LB
- Incubate on ice for 10 minutes
- Plate 50 μL and 250 μL onto the appropriate medium (e.g. LB-Sm-Nm)
Solutions
Mg2+/Ca2+
- 3.25 g MgCl2·6H2O
- 0.6 g CaCl2·2H2O
- 200 mL dH2O
Autoclave
100 mM Calcium Chloride
- 2.95 g CaCl2·2H2O
- 200 mL dH2O
Autoclave