Team:SCUT-China/Team/Protocol

From 2014.igem.org

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.