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Team:Hawaii/Protocols/Transformation E coli
From 2008.igem.org
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== Protocol == | == Protocol == | ||
# Thawed cells on ice. | # Thawed cells on ice. | ||
| - | # Transformed 50 μl cells w/ 1 μl | + | # Transformed 50 μl cells w/ 1 μl DNA |
# Incubated on ice 30 min. | # Incubated on ice 30 min. | ||
# Heat shocked 60 sec. in 42C water bath | # Heat shocked 60 sec. in 42C water bath | ||
# Added 250 μl SOC | # Added 250 μl SOC | ||
# Incubated @ 37C for 1 hour with shaking (150 rpm) | # Incubated @ 37C for 1 hour with shaking (150 rpm) | ||
| - | # Plated 20 μl onto LB+ | + | # Plated 20-250 μl onto LB+antibiotic plates |
:* Plate more if needed. | :* Plate more if needed. | ||
| + | ==Notes== | ||
| + | * Adjust DNA amount as necessary. Transformation is saturated at >10ng DNA. DNA should not exceed 10% of cell volume (Re: SC). | ||
| + | * Incubating 10 min. on ice after adding DNA and returning cells to ice for 2 min. after heat shock seems to improve transformation efficiency. ''-GK'' | ||
Latest revision as of 05:34, 13 August 2008
Protocol
- Thawed cells on ice.
- Transformed 50 μl cells w/ 1 μl DNA
- Incubated on ice 30 min.
- Heat shocked 60 sec. in 42C water bath
- Added 250 μl SOC
- Incubated @ 37C for 1 hour with shaking (150 rpm)
- Plated 20-250 μl onto LB+antibiotic plates
- Plate more if needed.
Notes
- Adjust DNA amount as necessary. Transformation is saturated at >10ng DNA. DNA should not exceed 10% of cell volume (Re: SC).
- Incubating 10 min. on ice after adding DNA and returning cells to ice for 2 min. after heat shock seems to improve transformation efficiency. -GK
