Difference between revisions of "Transformation of Bacteria"

From Bridges Lab Protocols
Jump to: navigation, search
m (Update)
(updated transformation protocol)
 
(One intermediate revision by the same user not shown)
Line 1: Line 1:
 
==Materials==
 
==Materials==
 
*Competent Cells
 
*Competent Cells
*Plasmid amplification use subcloning efficiency DH5a
+
*SOC Buffer or LB Media
*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)
 
*DNA of interest (1 uL for plasmid, 5 uL for cloning/mutagenesis)
*Plates (Amp or Kan; in cold room)
+
*Plates (Amp or Kan; in cold room, see [[ Making LB Agar Plates ]])
  
 
==Protocol==
 
==Protocol==
Line 14: Line 11:
 
#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 on shelf OR add 1000uL and adjust the level.
+
#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.

Latest revision as of 15:44, 12 February 2014

Materials

  • Competent Cells
  • SOC Buffer or LB Media
  • DNA of interest (1 uL for plasmid, 5 uL for cloning/mutagenesis)
  • Plates (Amp or Kan; in cold room, see Making LB Agar Plates )

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 or LB.
  7. Incubate at 37C for 1h with occasional mixing
  8. 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.