Difference between revisions of "Transformation of Bacteria"

Revision as of 22:48, 1 May 2009

Materials

• Competent Cells
• Plasmid amplification use subcloning efficiency DH5a
• Cloning use OneShot TOP10 (Pink)
• Mutagenesis use XL1 Blue Supercompetent (Blue)
• SOC Buffer
• DNA of interest (1 uL for plasmid, 5 uL for cloning/mutagenesis)
• Plates (Amp or Kan; in cold room)

Protocol

1. Thaw cells on ice and label
2. Add DNA to cells and mix by tapping
3. Incubate on ice 30-45min
4. Heat shock at 42C for 45s
5. Place back on ice
6. Add 450 uL of SOC Buffer
7. Incubate at 37C for 1h with occasional mixing
8. Plate 50 uL for amplification, or all for cloning/mutagenesis