Transformation of Bacteria
From Bridges Lab Protocols
Revision as of 18:56, 23 July 2012 by Davebridges (Talk | contribs) (removed confusing buffer addition)
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.
