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Team:Rice University/Heat Shock
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| - | WHAT YOU WILL NEED:<BR> | + | '''WHAT YOU WILL NEED:'''<BR> |
| - | ice bucket<BR> | + | ''ice bucket''<BR> |
| - | chemically competent cells - located in 1.5mL microcentrifuge tubes in -80*C freezer. Thaw in ice bucket (1 tube per transformation)<BR> | + | ''chemically competent cells'' - located in 1.5mL microcentrifuge tubes in -80*C freezer. Thaw in ice bucket (1 tube per transformation)<BR> |
| - | DNA - purified (purity is not as necessary as in electroporation)<BR> | + | ''DNA'' - purified (purity is not as necessary as in electroporation)<BR> |
| - | SOC media - recipe located in media folder. Prewarm to 42*C. (1mL per transformation)<BR> | + | ''SOC media'' - recipe located in media folder. Prewarm to 42*C. (1mL per transformation)<BR> |
| - | LB-agar antibiotic plate - located in 4*C far left. Make sure to get plate with appropriate antibiotic. Dry plate out in 37*C incubator at least 30 minutes before plating.<BR> | + | ''LB-agar antibiotic plate'' - located in 4*C far left. Make sure to get plate with appropriate antibiotic. Dry plate out in 37*C incubator at least 30 minutes before plating.<BR> |
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Revision as of 18:50, 18 June 2008
Chemical Transformation (Heat-Shock)
WHAT YOU WILL NEED:
ice bucket
chemically competent cells - located in 1.5mL microcentrifuge tubes in -80*C freezer. Thaw in ice bucket (1 tube per transformation)
DNA - purified (purity is not as necessary as in electroporation)
SOC media - recipe located in media folder. Prewarm to 42*C. (1mL per transformation)
LB-agar antibiotic plate - located in 4*C far left. Make sure to get plate with appropriate antibiotic. Dry plate out in 37*C incubator at least 30 minutes before plating.
1. Thaw the cells on ice (approx. 40uL).
2. Pippette DNA into tube containing cells and mix gently on ice. DO NOT CREATE BUBBLES IN THE CELLS. Temperature control is critical and bubbles heat them up. Usually 2uL of miniprepped DNA will be sufficient. If using less concentrated DNA (i.e. ligation product) up to 10 of DNA can be used.
3. Let DNA and cells incubate on ice for at least 30 min.
4. Heat pulse the cells at 42*C for exactly 45 sec. Time duration of heat pulse is critical.
5. Incubate cells on ice for 2 min.
6. Resuspend the cells in 1.0 mL of prewarmed 42*C SOC media. Incubate for 1 hour @ 37*C, shaking at at 250 rpm.
7. Plate in appropriate dilutions (in sterile 50% gylcerol) on LB-agar antibiotic plate using sterile glass beads. Goal is to obtain isolated colonies. If efficiency of transformation is unsure, plate multiple dilutions. Suggested dilutions: miniprepped DNA 5ul of 1/100,1/1,000, 1/10,000, ligation DNA >=20uL with no dilution.
