Exeter iGEM Lab Book

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.

05/06/15

• Digestion of our insert and backbone.
• Results of our transformations from our plates.

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