Team:TUDelft/ConjugationProtocol

From 2009.igem.org

Conjugation Protocol

Other Conjugation Protocols

This protocol was adapted from:

http://www.openwetware.org/wiki/Conjugation

and

http://openwetware.org/wiki/IGEM:Peking/2007/Count:Conjugation

TUDelft Conjugation Protocol

This is the Protocol Used by TUDelft iGEM 2009:

Day 1

  1. Get plates from fridge containing Donor and Recipient.
  2. Make 5 mL cultures with the appropriate antibiotics and leave overnight.

Day 2

  1. Prepare your plates (see tables at the bottom).
  2. Put 1 mL of the overnight cultures into 5 mL tubes. Usually 2 tubes of donors and 2 tubes of recipients.
  3. Test the OD600. You can use the following graph to estimate the time needed for E.coli containing R751 to reach an OD600 value of 0.45 to 0.6.
    R751 ODvsTIME.png
  4. Place 500 μL of culture into enough 1.5mL Eppendorfs for the conjugation test.
  5. Prepare AB free plates for the conjugation. One plate for each set.
  6. Centrifuge 1 at 3000 rcf for 3 min and discard supernatant.
  7. Resuspend in 500 μL LB without AB.
  8. Centrifuge 2 at 3000 rcf for 2 min and discard supernatant.
  9. Resuspend in 500 μL LB without AB.
  10. Centrifuge 3 at 3000 rcf for 1 min and discard supernatant.
  11. Resuspend in 500 μL LB without AB.
  12. Place cells on ice.
  13. Mix 100 μL of donors and 100 μL recipients in an eppendorf tube and vortex gently.
  14. Centrifuge for 1 min and discard supernatant.
  15. Resuspend the cell pellet in 10 μL of LB.
  16. Put a PALL 0.2 μM filter on top of a AB free plate. Gently pipette the conjugation mix on the filter. Use good sterile technique.
  17. Incubate for 1 h at 37ºC.
  18. Pick the filter from the plate with sterilized tweezers and put it into an 50 mL assay tube with 2 mL of LB broth.
  19. Vortex to re-suspend the cells.
  20. Make serial dilutions in LB broth. Plate in selective antibiotics for donors, recipients and transconjugants.
  21. The following plates are usually made with R = Recipient, D = Donor, and T = Transconjugant. Adjust the Antibiotics to those needed for your particular test.
Set 1
Plate ID Antibiotics Dilution
D1 TRI 100
D2 TRI 10-1
D3 TRI 10-2
D4 TRI 10-3
D5 TRI 10-4
T1 TRI + AMP 100
T2 TRI + AMP 10-1
T3 TRI + AMP 10-2
T4 TRI + AMP 10-3
T5 TRI + AMP 10-4
T6 TRI + AMP 10-5
T7 TRI + AMP 10-6
R1 AMP 100
R2 AMP 10-1
R3 AMP 10-2
R4 AMP 10-3



Set 2
Plate ID Antibiotics Dilution
D6 TRI 100
D7 TRI 10-1
D8 TRI 10-2
D9 TRI 10-3
D10 TRI 10-4
T8 TRI + AMP 100
T9 TRI + AMP 10-1
T10 TRI + AMP 10-2
T11 TRI + AMP 10-3
T12 TRI + AMP 10-4
T13 TRI + AMP 10-5
T14 TRI + AMP 10-6



Set 3
Plate ID Antibiotics Dilution
D11 TRI 100
D12 TRI 10-1
D13 TRI 10-2
D14 TRI 10-3
D15 TRI 10-4
T15 TRI + AMP 100
T16 TRI + AMP 10-1
T17 TRI + AMP 10-2
T18 TRI + AMP 10-3
T19 TRI + AMP 10-4
T20 TRI + AMP 10-5
T21 TRI + AMP 10-6

Day 3

Image your plates in the gel room.

For colonies with GFP use 50 ms exposure with the orange filter.

For colonies without GFP use 15 ms exposure with the upper white light turned on.

Retrieved from "http://2009.igem.org/Team:TUDelft/ConjugationProtocol"