IGEM:Peking/2007/Count:Conjugation
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(Difference between revisions)
| Line 10: | Line 10: | ||
===Donor=== | ===Donor=== | ||
| - | #Test OD600 after | + | #Test OD600 after 30 minutes of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase). |
#After sub-culturing decant the 500uL culture into a 1.5mL tube. | #After sub-culturing decant the 500uL culture into a 1.5mL tube. | ||
#spin down (top speed for 1 min), discard fluid. | #spin down (top speed for 1 min), discard fluid. | ||
| Line 26: | Line 26: | ||
#Place the cell suspension on ice. | #Place the cell suspension on ice. | ||
| - | === | + | ===mixing=== |
| + | *Donor cells(log phase, include control) mixed with recipient cells(stationary phase). | ||
| + | *Mix by vortexing. | ||
| + | *culture in 37℃ for 90 minutes,220rpm. | ||
| + | ===Plating=== | ||
| + | *Setting up serial dilutions: | ||
| + | *Pipette 200µL of each serial dilution onto an SFM+MgCl2 plate | ||
==see also== | ==see also== | ||
[[UC Berkeley/2006/Conjugation]] | [[UC Berkeley/2006/Conjugation]] | ||
Revision as of 07:00, 11 August 2007
Contents |
Conjugation Test
- choose the Donor cell and Recipient Cell which must be different Antibiotic.
*Day1:
- Get the plates from -4 fridge：Donor, Recipient, Control.
- Amplification Culture in liquid LB for 12 hours.
*day 2:
- put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.
Donor
- Test OD600 after 30 minutes of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase).
- After sub-culturing decant the 500uL culture into a 1.5mL tube.
- spin down (top speed for 1 min), discard fluid.
- resuspend in 500mL LB(NO Antibiotic!), vortex.
- Repeat steps 3-4-3 in this order then progress to step 6.
- Re-suspend with 500mL LB.
- Place the cell suspension on ice.
Recipient
- decant the recipient cells(500uL culture into a 1.5mL tube) when reached the stationary phase.
- spin down (top speed for 1 min.), discard fluid.
- resuspend in 500mL LB(NO Antibiotic!), vortex.
- Repeat steps 3-4-3 in this order then progress to step 6.
- Re-suspend with 500mL LB.
- Place the cell suspension on ice.
mixing
- Donor cells(log phase, include control) mixed with recipient cells(stationary phase).
- Mix by vortexing.
- culture in 37℃ for 90 minutes,220rpm.
Plating
- Setting up serial dilutions:
- Pipette 200µL of each serial dilution onto an SFM+MgCl2 plate
