Welcome to our lab book. The following pages contain a chronological description of everything we did in the lab – starting on Day 1. Different days contain short descriptions of our experiments and notes, as well as references to the appropriate protocols.
We were not adept at keeping a concise lab book during the first few weeks of iGEM – the descriptions get more organised and detailed as the weeks progress. The first part of our lab book relates to the Interlab Study, and the later parts to our actual project, Ribonostics.
All protocols can be found at the bottom of the page,and there will links throughout. If you have any questions you can email the team or individual members emails can be found on our team page.
04/06/15
Interlab Study - hydrating DNA, transformation into competent DH5α cells, overnight cultures, mini prep.
BioBrick construct DNA concentration (µg/mL)
Construct
I13504
J23108
Jasmine, Joe, Emilia (Star)
264.0
18.8
Amy, Yemi, Dan (Sharkisha)
314.0
23.6
Bradley, Georgina (Bob)
19.3
260.0
05/06/15
Digestion of our insert and backbone.
Results of our transformations from our plates.
Construct
Antibiotic
Colony number
Star
Cam
0
Cam
32
Amp
16
Amp
0
Sharkisha
Cam
29
Cam
6
Amp
0
Amp
15
Bob
Cam
36
Cam
50
Amp
0
Amp
7
08/06/15
Diagnostic digestion of previous ligations:
II3504 + J23108 = iGEMILS08A
Il3504 + J23108 = iGEMILS08B
Used EcoRI and SpeI during our digestion.
09/06/15
Diagnostic digestion of previous ligations.
Planned to miniprep iGEMILS08A AND iGEMILS08B, BUT we realised that we were using the wrong BioBricks all along - J23111, J23109, and J23108, INSTEAD of J23101, J23106 and J23117.
Resuspending DNA from Kit 1 (BioBricks J23101, J23106, J23117).
Hydrating DNA from the kit.
Poured plates necessary for our experiments.
Chloramphenicol plates – 450 µl chloramphenicol needed for bottle of LB broth of 450 mL.
Chloramphenicol plates poured out on the lab bench, not inside a flow hood. This can be done as they contain antibiotic – never do this with just LB broth (if it is just LB broth, use flow hood and aseptic technique).
Transformation (E. coli with J23101, J23106 and J23117).
Plates containing E. coli with plasmids J23101, J23106 and J23117, put in the 37°C static incubator (2 each – one with 20 µl of culture, one with 200 µl of culture).
Take out at 9 am on 10/06/15.
10/06/15
Plates taken out at 9 am and put in the cold room until 3 pm.
After 3 pm, they were taken out and overnight cultures were made. Transformation plates used:
J23117: 200 µl
J23101: 200 µl
J23106: 200 µl
Making overnight cultures.
11/06/15
Miniprep J23106A, B, C, J23117 A, B (C has been discarded due to possible contamination), J23101A, B, C.
Miniprep and ligation.
Names of newly ligated plasmids:
J23101 + I13504 = IGEMILS101
J23106 + I13504 = IGEMILS106
J23117 + I13504 = IGEMILS117
Making agarose gel for electrophoresis.
Making competent cells.
Qubit. Readings in table:
Plasmid
Concentration of DNA (ng/mL)
Stock concentration (ng/ µl)
J23101A
-
49.60
J23101B
-
2.54
J23101C
-
13.40
J23106A
1.360
154.00
J23106B
0.492
83.80
J23107C
0.118
87.20
J23117A
0.110
44.20
J23117B
0.189
72.80
I13504
0.962
103.00
Plasmid
Volume needed for digest (µl)
J23101A
5.00
J23101B
98.40
J23101C
18.60
J23106A
1.60
J23106B
3.00
J23107C
2.90
J23117A
5.70
J23117B
3.40
I13504
2.40
Plasmid
Water to be added (µl)
J23101A
11.0
J23106A
14.4
J23106B
13.0
J23106C
13.1
J23117A
10.3
J23117B
12.6
I13504
13.5
12/06/15
Autoclaving
15/06/15
Checked if colonies have been transformed (should glow green) – ILS101, ILS106, ILS117.
Overnight cultures of ILS101, ILS106, ILS117 (6 mL LB, 6 µl CAM, culture).
Glycerol stocks.
Pour CAM plates.
Miniprep ILS101.
overnight cultures 106, 117 and 108.
Diagnostic digestion.
16/06/15
Miniprep.
Digest.
Re-plating.
Glycerol stocks.
Qubit results:
Plasmid
Concentration (ng/mL)
ILS101A
Too low.
ILS101B
3.14
ILS101C
2.66
ILS101D
2.89
ILS101E
3.13
ILS106A
2.84
ILS106B
2.15
ILS106C
0.90
ILS106D
3.83
ILS106E
4.28
ILS117A
2.92
ILS117B
0.98
ILS117C
Too low.
ILS117D
1.24
ILS117E
1.12
Control Digest:
X1 (µl)
X10 (µl)
H2O
6.2
62
Buffer
1.0
10
EcoRI
0.4
4
Pstl
0.4
4
temp
2.0
-
total
10.0
8
Left to Right:
1kb ladder
101B
101C
101D
101E
106A
106B
106D
106E
117A
1kb ruler and ladder
22/06/15
Interlab study failed again
Start again with miniprepped J23101, J23106, J23117 and I13504
Do a standard digestion and a traditional digestion (1 µl of each enzyme, 1 µg of DNA, 2 µl green buffer, gel extraction – 1% agarose gel – run the whole sample)
