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
- 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
|