IGEM:Peking/2007/Count:Conjugation: Difference between revisions
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*choose the Donor cell and Recipient Cell which must be different Antibiotic. | *choose the Donor cell and Recipient Cell which must be different Antibiotic. | ||
== | ==Day1:== | ||
#Get the plates from -4 fridge:Donor, Recipient, Control. | #Get the plates from -4 fridge:Donor, Recipient, Control. | ||
#Amplification Culture in liquid LB for 12 hours. | #Amplification Culture in liquid LB for 12 hours. | ||
== | ==day 2:== | ||
#put 2mL of culture into 20mL of LB with antibiotics, sub-culturing. | #put 2mL of culture into 20mL of LB with antibiotics, sub-culturing. | ||
Line 29: | Line 29: | ||
*Donor cells(log phase, include control) mixed with recipient cells(stationary phase). | *Donor cells(log phase, include control) mixed with recipient cells(stationary phase). | ||
*Mix by vortexing. | *Mix by vortexing. | ||
*culture in 37℃ for 90 minutes,220rpm. | *culture in 37℃ for 90 minutes, 220rpm. | ||
===Plating=== | ===Plating=== | ||
*Setting up serial dilutions: | *Setting up serial dilutions:original, 10-1, 10-2. | ||
*Pipette 200µL of each serial dilution onto an | *Pipette 200µL of each serial dilution onto an Double antibiotic plate. | ||
===result=== | |||
*The frequency of transfer was expressed as the ratio (percentage) of the number of transconjugants to that of the donors. | |||
==see also== | ==see also== | ||
[[IGEM:UC_Berkeley/2006/Conjugation]] | [[IGEM:UC_Berkeley/2006/Conjugation]] |
Revision as of 04:19, 11 August 2007
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:original, 10-1, 10-2.
- Pipette 200µL of each serial dilution onto an Double antibiotic plate.
result
- The frequency of transfer was expressed as the ratio (percentage) of the number of transconjugants to that of the donors.