Lab Notebook
From 2006.igem.org
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Regarding the MSCl, the seeding from the 19th did not work, both the controls were found to be murky (ampicillin and LB as well as LB only) therefore this was performed again. | Regarding the MSCl, the seeding from the 19th did not work, both the controls were found to be murky (ampicillin and LB as well as LB only) therefore this was performed again. | ||
| - | '''May 24''': | + | '''May 24''': 4 or 5 colonies on plates. Seeded 3 colonies in 3 tubes with 5mL LB. [Initially believed there were no colonies and ligation didn't work. We ran a gel of the ligation to see if it contained the Luciferase gene or any DNA at all (since the extraction procedure might not have worked).] Miniprep of Stable 3 bacteria with pB10 vector containing MscL was performed. |
Revision as of 03:13, 25 May 2006
May 15: Transformed Top10F' bacteria with the pGFP vector containing the Luciferase gene (hopefully the ligation worked this time).17:22, 15 May 2006 (EDT)
May 16: Transformation of Top10F did not work. The cause was an error in the extraction of the DNA from the the agarose gel. Completed a new agarose gel extraction of pGFP plasmid DNA, as cut by EcoRI and HindIII. Prepared a new seed of Top10F bacteria transformed with luciferase. Prepared a midiprep seed of EcoMscL.
May 17: The control of the miniprep? of luciferase was murky. This could have been due to contaminants in the LB, water, ampicillin, or pipet tips. A miniprep of both luciferase and EcoMscL was conducted. A new seed of luciferase was prepared. The miniprep of luciferase was digested with EcoRI and HindIII. The miniprep of EcoMscL was digested with XmnI. A gel with the digests was run but the EcoMscL showed no bands and the luciferase only had faint bands.
May 18: The controls of the seed of luciferase were clear. A miniprep of the new luciferase seed and the old EcoMscL seed was conducted. The same restriction enzymes were used to digest the plasmids. A gel was run with the digests. There were two faint bands on the gel with the EcoMscL when there should have been three. Thus a new batch of Stable 3 bacteria were transformed with EcoMscL through heat shock and plated. The gel with the psp-Luciferase turned out well and the gene was extracted from the gel. Ligation was not completed since the microcentrifuge tube with the pGFP cut vector could not be found due to labelling issues.
May 19: The Stable3 plates were good. We Seeded with 5 microliters of Amp in 2.5mL of LB and 2.5 mL of sterile water and placed in shaker-incubator overnight. We also ligated pGFP & Luciferase (+2 controls, c.f. lab manual) incubating at room temp for 1h. The ligation was then put in the freezer.
May 21: Put up an example of cloning for oscillator.
May 23: A transformation of the luc pGFP ligation product was performed. It was reasoned not to do transfomation on the control (with no ligase) in order to save Top Ten colonies. Also only the transformation of only the ligation with 12 microliters luciferase and 3 microliters pGFP was done, not the 6 microliter luciferase one for the same reason as mentioned above. Only half (10 microliters) of the 12 uL-ligation was used, the rest stored in freezer. The result was plated. Regarding the MSCl, the seeding from the 19th did not work, both the controls were found to be murky (ampicillin and LB as well as LB only) therefore this was performed again.
May 24: 4 or 5 colonies on plates. Seeded 3 colonies in 3 tubes with 5mL LB. [Initially believed there were no colonies and ligation didn't work. We ran a gel of the ligation to see if it contained the Luciferase gene or any DNA at all (since the extraction procedure might not have worked).] Miniprep of Stable 3 bacteria with pB10 vector containing MscL was performed.
