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Revision as of 18:52, 29 October 2017
Lab-Book </h1>
19/07/2017
Media Preparation:
LB agar Miller: 4 x 200 mL bottles
- Add 8 g of agar in scott bottle.
- Pour in 200 mL of distilled water.
- Gently shake.
- Autoclave at
- Store at room temperature.
LB broth Miller:
2 x 25 g in 1 L.
5 x 50 mL in conical flasks.
50 x 10 ml in universal flasks.
24/07/17
Media Preparation (had to remake the Agar)
8x(200ml dH2O + 8g LB Agar).
25/07/17
Cultivating E.coli part 1
- Pouring out agar plates..
- Streaking plates with E.coli..
- Put plates in incubation at 37’C for 15 hours max (put in at 6.08pm.).
26/07/17
Cultivating E.coli part 2 (Round 1)
- Pick single colony of the cells from the LB agar plate into 10 ml of LB media containing no antibiotics OR specific for cell type..
- Grew the cultures overnight at 37’C with shaking incubator at 250 rpm..
Pre pouring reagents for competent cells
- 0.1 M MgCl2 solution: 2.033g of MgCl2 6H2O in 100 ml H20 in a 100ml Pyrex Bottle..
- 0.1 M CaCl2 solution: 11g of Cacl2 in 500ml H20 in a 500ml Pyrex Bottle..
- 50% Glycerol solution: 10 ml of glycerol & 10ml H20 in 20ml culture tube with screw cup, autoclave at 4’C..
27/07/2017
Competent cells (Round 1)
- Inoculate 200 mL of pre-warmed to 37 degrees C LB medium (w/out antibiotics) with 10 mL of overnight E.coli cultures..
- Incubate at 37 degrees C for 60 min with vigorous shaking 250 rpm or until OD600 is 0.4-0.5..
- Put flask on ice for 30 min. At the same time chill sterile falcon tubes..
- Aliquot culture into 50 mL each falcon tube (4x50).
- Harvest cells by centrifugation for 7 min at 3,500 rpm at 4 degrees C and discard supernatant.
- Resuspend cells with 12.5mL of MgCl2.
- Centrifuge for 7 min at 3,500rpm at 4 degrees C and discard supernatant.
- Resuspend cells with 25 mL of CaCl2 then incubate on ice for 30 min..
- Centrifuge for 7min at 3,500rpm at 4 degrees C and discard supernatant.
- Resuspend cells with 700 microL of CaCl2 and 300 microL of 50% glycerol. Final volume 1 mL..
- Aliquot 50 microL aliquots into 1.5mL sterile eppendorf tubes and store at -80 degrees C..
Table of measured OD (during step 2):
Time (min)
OD600 batch 1
OD600 batch 2
0
0
0
15
0.043
0.047
30
0.031
0.012
45
0.065
0.005
60
0.108
/
75
0.150
/
90
0.422
/
NB: Calibrate with LB before inoculation, not with water.
Kanamycin plates
Cultivating E.coli part 1
Cultivating E.coli part 2 (Round 1)
Pre pouring reagents for competent cells
Competent cells (Round 1)
Table of measured OD (during step 2):
Time (min) |
OD600 batch 1 |
OD600 batch 2 |
0 |
0 |
0 |
15 |
0.043 |
0.047 |
30 |
0.031 |
0.012 |
45 |
0.065 |
0.005 |
60 |
0.108 |
/ |
75 |
0.150 |
/ |
90 |
0.422 |
/ |
Kanamycin plates
MgCl2
Assaying Competency of E.coli competent cells (Round 1) - failed attempt of transformation
LB Broth:
Stock Solution of DNA
Cultivating E.coli part 2 (Round 2)
Transformation (Round 1)
Transformation of E.coli competent cells made on 27/07/17 with Dr Sarah Coleman.<p>
Tubes |
1 |
2 |
3 |
4 |
5 |
Volume of DNA ul |
2 |
10 |
2 |
10 |
N/A |
Origin DNA |
IGEM |
IGEM |
Sarah |
Sarah |
Sarah |
OTE: No growth at all: 1) the cells were not competent, 2) PLates have too much antibiotic or wrong antibiotic.
Biofilm culture part 1 (Round 1)
Competent cell making (Round 2)
Biofilm culture part 2 (Round 1)
Transformation (Round 2)
Biofilm culture part 3 (Round 1)
Biofilm Culture part 3 (Round 1)
OD readings of flat-bottom plate: 1 2 3 4 C 1.31 1.21 1.67 1.407
Biofilm Culture part 1 (Round 2)
Transformation (Round 3):
09/08/17To assay biolfims - best use ‘endpoint’ setting in spectrometer. It would seem that the best time for CV incubation is 10 min. Both dyes present fairly similar values however, the mixed acetic acid/CV solution has highest OD.