IGEM:UC Berkeley/2006/Knockouts: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
JCAnderson (talk | contribs) No edit summary |
||
| Line 1: | Line 1: | ||
==Procedure for Knockouts== | |||
#50mL LB+Carb+Kan and 5mL starter culture plus 200mg/mL Arabinose | #50mL LB+Carb+Kan and 5mL starter culture plus 200mg/mL Arabinose | ||
#Grow to OD=.8 (the higher the better) | #Grow to OD=.8 (the higher the better) | ||
| Line 10: | Line 10: | ||
#Rescue one hour @ 37C | #Rescue one hour @ 37C | ||
#Plate | #Plate | ||
==Excising FRT-flanked cassettes with pCP20== | |||
#Grow up a colony of the strain containing the cassette from a freshly restreaked plate | |||
#Prepare a small scale electrocompetent cell prep, transform with pCP20 | |||
#Plate on Amp, or Cm+Amp, at 30 degrees | |||
#Next day, use a tip or toothpick to swab some of the colonies, put in 4 mL of 2YT | |||
#Grow to saturation at 37 degrees | |||
#Dilute 10^6, plate on LB-only plates at 43 degrees | |||
#Grow up and spot individual colonies on Amp and the excision marker | |||
Latest revision as of 11:35, 14 September 2006
Procedure for Knockouts
- 50mL LB+Carb+Kan and 5mL starter culture plus 200mg/mL Arabinose
- Grow to OD=.8 (the higher the better)
- Spin approx. 40mL in 50mL conical, 3 minutes @ 8000
- Resuspend 30mL 10% glycerol
- Spin 3 minutes @ 7500
- Repeat glycerol suspension (step 4)
- Dump supernatant, resuspend in remaining liquid (don't add anything in this step)
- Electroporate 50ul cells and 1ul PCR product
- Rescue one hour @ 37C
- Plate
Excising FRT-flanked cassettes with pCP20
- Grow up a colony of the strain containing the cassette from a freshly restreaked plate
- Prepare a small scale electrocompetent cell prep, transform with pCP20
- Plate on Amp, or Cm+Amp, at 30 degrees
- Next day, use a tip or toothpick to swab some of the colonies, put in 4 mL of 2YT
- Grow to saturation at 37 degrees
- Dilute 10^6, plate on LB-only plates at 43 degrees
- Grow up and spot individual colonies on Amp and the excision marker
