Difference between revisions of "Transformation of Bacteria"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (copied over protocol) |
m (Update) |
||
| Line 3: | Line 3: | ||
*Plasmid amplification use subcloning efficiency DH5a | *Plasmid amplification use subcloning efficiency DH5a | ||
*Cloning use OneShot TOP10 (Pink) | *Cloning use OneShot TOP10 (Pink) | ||
| − | *Mutagenesis use XL1 Blue Supercompetent (Blue) | + | *Mutagenesis use XL1 Blue Supercompetent (Blue), comes in 50uL aliquots |
*SOC Buffer | *SOC Buffer | ||
*DNA of interest (1 uL for plasmid, 5 uL for cloning/mutagenesis) | *DNA of interest (1 uL for plasmid, 5 uL for cloning/mutagenesis) | ||
| Line 14: | Line 14: | ||
#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 | + | #Add 450 uL of SOC Buffer or LB on shelf OR add 1000uL and adjust the level. |
#Incubate at 37C for 1h with occasional mixing | #Incubate at 37C for 1h with occasional mixing | ||
| − | #Plate 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. | ||
Revision as of 15:05, 5 May 2009
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 on shelf OR add 1000uL and adjust the level.
- 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.
