Griffitts:Chemically competent cells: Difference between revisions
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==Procedure== | ==Procedure== | ||
* Grow DH5α overnight in 5 mL [[Griffitts:Common media#LB Broth (1 L)|LB]] at 37°C | * Grow DH5α (B095) overnight in 5 mL [[Griffitts:Common media#LB Broth (1 L)|LB]] at 37°C | ||
* Inoculate 100 mL [[Griffitts:Common media#LB Broth (1 L)|LB]] with 1 mL of saturated overnight culture of DH5α | * Inoculate 100 mL [[Griffitts:Common media#LB Broth (1 L)|LB]] with 1 mL of saturated overnight culture of DH5α | ||
* Shake at 37°C until OD<sub>600</sub> = 0.4 | * Shake at 37°C until OD<sub>600</sub> = 0.4 | ||
** Usually 2 | ** Usually 2–3 hours | ||
* Place in an ice bath for 5 minutes | * Place in an ice bath for 5 minutes | ||
Note: After this point the cells should never touch anything that is warm—chill solutions, pipets, tubes, etc. beforehand<br> | Note: After this point the cells should never touch anything that is warm—chill solutions, pipets, tubes, etc. beforehand<br> | ||
Line 18: | Line 20: | ||
* Add 0.5 mL cold 80% glycerol and swirl to mix | * Add 0.5 mL cold 80% glycerol and swirl to mix | ||
* Flash-freeze in liquid nitrogen as 100-μL aliquots | * Flash-freeze in liquid nitrogen as 100-μL aliquots | ||
* Store in the | * Store in the –80°C freezer | ||
==Transformation== | ==Transformation== | ||
* Add | * Add 7 μL ligation mix to [[Griffitts:Chemocompetent Cells#Procedure|chemocompetent DH5α cells]] | ||
* Incubate on ice for | * Incubate on ice for 5–10 minutes | ||
* Heat shock at 42°C for 1 | * Heat shock at 42°C for 1 minute | ||
* Add 800 μL | * Incubate on ice for 2 minutes | ||
* | * Add 800 μL [[Griffitts:Common media#LB Broth (1 L)|LB]] | ||
* Plate 50 μL and 250 μL onto the appropriate medium | * Tape to a shaker at the appropriate temperature for 1 hr. | ||
* Plate 50 μL and 250 μL onto the appropriate selective medium | |||
==Solutions== | ==Solutions== | ||
===Mg<sup>2+</sup>/Ca<sup>2+</sup>=== | ===Mg<sup>2+</sup>/Ca<sup>2+</sup>=== | ||
Line 37: | Line 41: | ||
* 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<br> | ||
Note: You can also make a 1:10 dilution of the [[Griffitts:Stock solutions#1 M Calcium Chloride (75 mL)|1 M stock]] |
Latest revision as of 15:29, 29 April 2009
Procedure
- Grow DH5α (B095) 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
Note: After this point the cells should never touch anything that is warm—chill solutions, pipets, tubes, etc. beforehand
- 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 7 μL ligation mix to chemocompetent DH5α cells
- Incubate on ice for 5–10 minutes
- Heat shock at 42°C for 1 minute
- Incubate on ice for 2 minutes
- Add 800 μL LB
- Tape to a shaker at the appropriate temperature for 1 hr.
- Plate 50 μL and 250 μL onto the appropriate selective medium
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
Note: You can also make a 1:10 dilution of the 1 M stock