Transformation of Bacteria: Difference between revisions
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*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) | ||
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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 | #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. | |||