IGEM:Peking/2007/Count:Conjugation

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===Donor===
===Donor===
-
#Test OD600 after 30minutes of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase).
+
#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.
-
===Plating the conjugant mix ===
+
===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:

  1. Get the plates from -4 fridge:Donor, Recipient, Control.
  2. Amplification Culture in liquid LB for 12 hours.

*day 2:

  1. put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.

Donor

  1. Test OD600 after 30 minutes of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase).
  2. After sub-culturing decant the 500uL culture into a 1.5mL tube.
  3. spin down (top speed for 1 min), discard fluid.
  4. resuspend in 500mL LB(NO Antibiotic!), vortex.
  5. Repeat steps 3-4-3 in this order then progress to step 6.
  6. Re-suspend with 500mL LB.
  7. Place the cell suspension on ice.

Recipient

  1. decant the recipient cells(500uL culture into a 1.5mL tube) when reached the stationary phase.
  2. spin down (top speed for 1 min.), discard fluid.
  3. resuspend in 500mL LB(NO Antibiotic!), vortex.
  4. Repeat steps 3-4-3 in this order then progress to step 6.
  5. Re-suspend with 500mL LB.
  6. 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

UC Berkeley/2006/Conjugation

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