Team:SCUT-China/Team/Protocol

CaCl2 competent cell

Materials

LB medium,Spectrophotometer,Shaking incubator at 37℃,Ice-cold CaCl₂,15% glycerol,Icebox,-80℃ refrigerator

Protocol

• Inoculate 5ml LB with E.coli; incubate over night at 37℃ and 250 rpm.
• Inoculate 100ml LB with an aliquot of an overnight culture and grow to an O.D.600 of 0.3-0.4 at 37℃ with 250 rpm.
• Divide the cultures into two 50ml Epppendorf tubes.
• Keep on ice for 30min.
• Centrifuged at 4000rpm for 4min at 4℃.
• Discard supernatant.
• Resuspended in 10ml ice-cold CaCl₂.
• Keeping on ice for 10min.
• Centrifuged at 4000rpm for 4min at 4 ℃ again.
• Discard supernatant.
• Resuspended in 10ml ice-cold CaCl₂.
• Keeping on ice for 10min.
• Repeat the step 5-8 again.
• Centrifuged at 4000rpm for 4min at 4 ℃.
• Discard supernatant.
• Resuspended in 2ml CaCl₂ containing 15% glycerol.
• Devide it into 1.5ml Eppendorf tubes, 100ul per tube.
• Stored at -80℃.

Heat-shock transformation
Materials
Plasmid or DNA ligation mix,Water bath 42℃,LB medium,Shaking incubator at 37℃,Eppendorf centrifugation,Selective plate

Protocol

• Add 1-2ul plasmid to 100ul competent cells. Homogenize by gently mixing with pipette several times. (Note that if the plasmid is product of linkage, add 10-20ul DNA)
• Keep on ice for 30min.
Retrieved from "http://2014.igem.org/Team:SCUT-China/Team/Protocol"