Difference between revisions of "Transformation of Bacteria"
From Bridges Lab Protocols
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Davebridges (Talk | contribs) (removed confusing buffer addition) |
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#Heat shock at 42C for 45s | #Heat shock at 42C for 45s | ||
#Place back on ice | #Place back on ice | ||
| − | #Add 450 uL of SOC Buffer or LB | + | #Add 450 uL of SOC Buffer or LB. |
#Incubate at 37C for 1h with occasional mixing | #Incubate at 37C for 1h with occasional mixing | ||
#Plate 30-50 uL for amplification, or all for cloning/mutagenesis | #Plate 30-50 uL for amplification, or all for cloning/mutagenesis | ||
*When plating be sure to label plates and use extreme caution when sterilizing the spreader with EtOH and fire. | *When plating be sure to label plates and use extreme caution when sterilizing the spreader with EtOH and fire. | ||
Revision as of 18:56, 23 July 2012
Materials
- Competent Cells
- Plasmid amplification use subcloning efficiency DH5a
- Cloning use OneShot TOP10 (Pink)
- Mutagenesis use XL1 Blue Supercompetent (Blue), comes in 50uL aliquots
- SOC Buffer
- DNA of interest (1 uL for plasmid, 5 uL for cloning/mutagenesis)
- Plates (Amp or Kan; in cold room)
Protocol
- Thaw cells on ice and label
- Add DNA to cells and mix by tapping
- Incubate on ice 30-45min
- Heat shock at 42C for 45s
- Place back on ice
- Add 450 uL of SOC Buffer or LB.
- Incubate at 37C for 1h with occasional mixing
- Plate 30-50 uL for amplification, or all for cloning/mutagenesis
- When plating be sure to label plates and use extreme caution when sterilizing the spreader with EtOH and fire.
