IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/05/29

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Testing of Chloramphenicol

  • This time round, the LB-Chloramphenicol was prepared by adding 40µL of 1000X Chloramphenicol stock (25 mg/mL) into 40 mL of LB-Agar.
  • The plates were poured the day before, and today they were streaked. Each LB-only and LB-Chlor plate had both DB3.1 and DH5alpha cells.
  • They were left on Roza's bench over the weekend for growth; will check back on these on Monday.

Restriction Digest of Amp Construction Plasmid, GFP, J23100+RBS, Terminator and pBAD

  • A master mix of 30uL 10X Buffer, 228uL of water and 6uL of EcoRI restriction enzyme was made
  • 44uL of the master mix was pipetted into 6uL of plasmid DNA
  • The solution was stored in the 37°C incubator for 2.5 hours
  • 6ul of DNA ladder and 25uL of each solution was loaded into the gel box

Transformation of New DH5α, BW27783, and test plasmid with old DH5α and old DB3.1

  • 1uL of the pNHG1 plasmid containing a Kan and a Tet resistance gene was pipetted into each of the 100uL of the 09/05/21 DH5α cells and 100uL of 09/05/21 DB3.1 cells as a control for the Kan and Tet construction plasmids
  • 1uL of the pMMB207 plasmid containing a Amp resistance gene was pipetted into each of the 100uL of the 09/05/29 DH5α cells and 100uL 09/05/29 BW27783 cells to check for compentency
  • The cells were kept on ice for 30 min
  • Heat shock for 60sec at 42°C
  • Put on ice for 2 min
  • 400ul of LB was added to each of the samples
  • Incubated for 1h 45min at 37°C
  • 250uL each of 09/05/21 DH5α+pNHG1 was plated onto a Kan and a Tet plate
  • 250uL each of 09/05/21 DB3.1+pNHG1 was plated onto a Kan and a Tet plate
  • 500uL of 09/05/29 DH5α+pMMB207 was plated onto an Amp plate
  • 500uL of BW27783+pMMB207 was plated onto an Amp plate