Melbourne/Lab Notebook
From 2007.igem.org
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*[[Melbourne/BBa_E0241|'''P2 15L 1''']] | *[[Melbourne/BBa_E0241|'''P2 15L 1''']] | ||
*[[Melbourne/BBa_E0241|'''P2 15L 2''']] | *[[Melbourne/BBa_E0241|'''P2 15L 2''']] | ||
| - | + | *[[Melbourne/BBa_Q04510|'''P2 13K 1''']] | |
| - | + | *[[Melbourne/BBa_Q04510|'''P2 13K 2''']] | |
=====EcoRI/HaeII in buffer 2===== | =====EcoRI/HaeII in buffer 2===== | ||
*[[Melbourne/BBa_B0010|'''P2 3P 1''']] | *[[Melbourne/BBa_B0010|'''P2 3P 1''']] | ||
| Line 179: | Line 179: | ||
===6 July 2007=== | ===6 July 2007=== | ||
| + | |||
| + | ====Digest Gel==== | ||
| + | * Prepared 20 lane 100mL [[Melbourne/Preparing an agarose gel|agarose gel]] with 0.5xTBE buffer. | ||
| + | *Loaded 20uL of digest samples in the following lane order | ||
| + | *#1kb+ ladder | ||
==Week 3== | ==Week 3== | ||
Revision as of 09:06, 8 July 2007
Contents |
Week 1
- 25 June 2007: Prepared LB agar plates Amp & Kana.
- 25 June 2007: Resuspended the following from Registry plates & Transformed (shorter protocol) into competent DH5alpha cells.
- 26 June 2007: Resuspended the following from Registry plates & Transformed into Joe's competent DH5alpha cells.
- 26 June 2007: Streaked the following cells:
- pJS010 (from solid agar, Amp)
- Fusion protein (from glycerol stock, Amp?)
- BBa_I15010 (from solid agar, Kan)
- 27 June 2007: Transformed into Joe's competent DH5alpha cells.
Liquid culture
- Prepared 30mL of Amp LB (100ug/mL). Prepared 5mL Kan LB (50mg/mL).
- Aliquoted 5mL Amp LB into 6 50mL falcon tubes
- To the Amp LB aliquots single transformed colonies from plates of the following were introduced:
- To the Kan LB a single colony from the transformation plate of BBa_I15010 was introduced.
- Cells incubated at 37degrees with shaking overnight.
28 June 2007
Miniprep
- Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
- Stored in -20 freezer
Digest
Liquid culture
- Cultured the following cells from transformed plates:
29 June 2007
Miniprep
- Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
- Stored in -20 freezer
30 June 2007
Week 2
- 2 July 2007: Resuspended the following from Registry plates & Transformed into Joe's competent DH5alpha cells.
- Q04510
- E0241
- E0040
- B0014
- J61035
- 3 July 2007: Re Transformed into Joe's competent DH5alpha cells. Each Plate had additional 40uL of 50mg/ml Ampicillan spread on surface.
- 4 July 2007:
Ampicillin Plates
- Made up 25 Ampicillin plates using 400mL LB agar prepared on the 25th of June.
- Used 100mg/ml Ampicillin stock from Gooley Lab - In bag in left freezer near our lab bench.
Tranformation
Transformed the following and grew on new ampicillin plates
- P1 5H
- P4 8J
- P2 15L
- Also Plated an untransformed control (subjected to transformation protocol in absense of DNA) and a blank plate control (no cells)
Liquid Culture
- Cultured 2 colonies from each of the following transformed plates and labelled as follows
5 July 2007
Miniprep
- miniprepped the following overnight cultures set up on the 4th of July. Final elution was performed with TE buffer prepared on 25/06/07 rather than nuclease free water.
- the following liquid cultures were not miniprepped due to failure (no growth)
Digest
Performed the following digests on DNA from the above miniprep
EcoRI/PstI with buffer 3
EcoRI/HaeII in buffer 2
XbaI/SpeI in buffer 2
Incubated for 2hours at 37 degrees, before addition of loading dye and storage at -20
Transformation
- Transformed P1 11H from resuspended DNA.
6 July 2007
Digest Gel
- Prepared 20 lane 100mL agarose gel with 0.5xTBE buffer.
- Loaded 20uL of digest samples in the following lane order
- 1kb+ ladder
