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+),...)
Current revision (02:23, 1 September 2007) (view source)
(Plating)
 
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==Conjugation Test==
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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:'''
+
==Day1:==
-
#Get the plates from -4 fridge:C600-R751_psc101(Tc+), Dh5α_psb1A2(Amp+), C600_R751, Dh5α_psc101
+
#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:'''
+
 
 +
==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===
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#Test OD600 after 30minutes of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase).
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#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.
#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.
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#Place the cell suspension on ice.
#Place the cell suspension on ice.
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===Plating the conjugant mix ===
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===mixing===
 +
*Donor cells(log phase, include control) mixed with recipient cells(stationary phase).
 +
*Mix by vortexing.
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*culture in 37℃ for 90 minutes, 220rpm.
 +
 
 +
===Plating===
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*Setting up serial dilutions:original, 10-1, 10-2, 10-3, 10-4, 10-5
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*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.
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===result===
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==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:

  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: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

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