IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/05/22

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Testing of LB-Kan Plates

  • Tested LB-Kan plates by streaking frozen glycerol stocks of DB3.1 cells on LB plate and LB-Kan plate.
  • Expect growth on LB plate and no growth on LB-Kan plate.
  • Incubated at 37 deg C in the Lagally Lab Biohazard room at 11AM.

Transformation and Calculation of Cell Competency

Number of Colonies
DH5a DB3.1
100uL 0 2
400uL 29 25
Competency 3.6*106cfu/μg 3.1*106cfu/μg

Example calculation:
1uL of 10pg/uL pUC19 pDNA + 100uL competent cells + 400uL LB recovery media
400uL plates are 8*10-12g giving rise to 29 or 25 colonies.
29cfu/(8*10-6μg) = 3.6*106cfu/μg


Made Chloramphenicol

  • 25mg/mL stocks; 20 aliquots of 1 mL each, therefore 20 mL
  • Dissolved 500 mg (0.5 g) of Chloramphenicol into 20 mL of 100% Ethanol
  • Aliquoted 1 mL ea. into sterile microfuge tubes, freezed at -20ºC

Extracted BioBricks from the wells

  • Resuspended the BioBricks from inside their wells with 15μL diH2O
  • Extracted the BioBricks into microfuge tubes


BioBricks Extracted:
Plate/Well Antibiotics
GRP_LVA 2 / 2L Amp
Terminator 2 / 24C Amp/Kan
pBAD 1 / 1F Amp
J23100+RBS 1 / 5J Amp
Construction (Amp) 1 / 3K Amp
Construction (Chl) 1 / 5E Chl
Construction (Kan) 1 / 7I Kan
Construction (Tet) 1 / 9E Tet

Transformation of Biobricks

Biobrick Plasmid Competent Cell Strain
GFP_LVA DH5a
Terminator DH5a
pBAD DH5a
J23100+RBS DH5a
Construction Plasmid(amp) DB3.1
Construction Plasmid(chl) DB3.1
Construction Plasmid(kan) DB3.1
Construction Plasmid(tet) DB3.1
  • Amount plated = 500uL
  • Amount of biobrick transformed = 1uL
  • amount of competent cell = 100uL of each.

Notes:

  • hot water bath was hotter than normal: 46ºC rather than 42ºC.
  • incubated for: 1 hour and 15 minutes(3:45pm to 5:00pm) @ 31ºC.
  • Tetracycline plate was left out in the light for about a hour. May cause degradation of tetracycline.
  • Incubating over the weekend (Friday night to Monday morning) at room temperature