IGEM:Peking/2007/Count:Conjugation
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< IGEM:Peking | 2007(Difference between revisions)
(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+),...) |
Current revision (02:23, 1 September 2007) (view source) (→Plating) |
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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]] | ||
Current revision
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: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.
