IGEM:Peking/2007/Count:Conjugation: Difference between revisions
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Qu Mingzhi (talk | contribs) (New page: ==Conjugation Test== *choose the Donor cell and Recipient Cell which must be different Antibiotic. '''*Day1:''' #Get the plates from -4 fridge:C600-R751_psc101(Tc+), Dh5α_psb1A2(Amp+),...) |
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=Conjugation Test= | |||
*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 | #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. | ||
===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 | #spin down (top speed for 1 min), discard fluid. | ||
#resuspend in 500mL LB(NO Antibiotic!), vortex. | #resuspend in 500mL LB(NO Antibiotic!), vortex. | ||
#Repeat steps 3-4-3 in this order then progress to step 6. | #Repeat steps 3-4-3 in this order then progress to step 6. | ||
#Re-suspend with 500mL LB. | #Re-suspend with 500mL LB. | ||
#Place the cell suspension on ice. | #Place the cell suspension on ice. | ||
===Recipient=== | ===Recipient=== | ||
#decant the recipient cells(500uL culture into a 1.5mL tube) when reached the stationary phase. | #decant the recipient cells(500uL culture into a 1.5mL tube) when reached the stationary phase. | ||
Line 23: | Line 26: | ||
#Place the cell suspension on ice. | #Place the cell suspension on ice. | ||
===Plating | ===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, 10-3, 10-4, 10-5 | |||
*Pipette 200µL of each serial dilution onto an Double antibiotic plate. | |||
*Notes: Some controls may growth on double antibiotic plate when these cultures are not being diluted. | |||
=== | ==result== | ||
*The frequency of transfer was expressed as the ratio (percentage) of the number of transconjugants to that of the donors. | |||
==see also== | |||
[[IGEM:UC_Berkeley/2006/Conjugation]] |
Latest revision as of 23:23, 31 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, 10-3, 10-4, 10-5
- Pipette 200µL of each serial dilution onto an Double antibiotic plate.
- Notes: Some controls may growth on double antibiotic plate when these cultures are not being diluted.
result
- The frequency of transfer was expressed as the ratio (percentage) of the number of transconjugants to that of the donors.