IGEM:Peking/2007/Count:Conjugation
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(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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| Line 1: | Line 1: | ||
| - | + | =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 30minutes of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase). | #Test OD600 after 30minutes 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 25: | Line 28: | ||
===Plating the conjugant mix === | ===Plating the conjugant mix === | ||
| - | == | + | ==see also== |
| + | [[UC Berkeley/2006/Conjugation]] | ||
Revision as of 06:39, 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 30minutes 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.
