Uppsala-Sweden/4 September 2009

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Revision as of 22:45, 5 September 2009 by Karl.brune (Talk | contribs)

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Glycerol Stock B0034-Yadh2

800ml ON culture + 200µl 87% glycerol @-80C rack 10 on pSB1AK3

--Karl.brune 12:17, 4 September 2009 (UTC)

Assembling, Transforming and and and

Digestion

Digestion was performed according to this file: Media:2009_09_04_Digestion.xls

Purification

Purification was performed for all samples with NucleoSpin2 form MN/Clontech. Before eluting the DNA I incubated the tubes for 5min at 70°C to let the ethanol evaporate -> better downstream performance.

DNA Measurements

DNA concentration were measured.

Results can be found in a nicer overview (link on pbworks), that hopefully everyone is going to use from now on ;) However here is the old school version: Media:2009_09_04_purified_digest_siRNA_and_parts.pdf

Ligation

Ligation was performed according to these files.

Media:2009_09_04_A3_Po_Ligate_into_Vector.xls

Media:2009_09_04_AC3_Po_Ligate_into_Vector.xls

Media:2009_09_04_AK3_Assembly_Ligation.xls

Media:2009_09_04_AC3_Assembly Ligation.xls

NEB quick ligation protocol was used.

Transformation

Competent TOP10 cells were thawn on ice for 30min 2µl of ligation product were put on top of the cells and the mix was stirred gently. After that I kept the cells on ice for aprox. 30min, followed by a 50sec. 42°C heatshock and 30min recovery on ice. Added 200µl of SOC, incubation @ 37°C 250RPM for 30min. Plating (200µl) on selective plates. Incubation at 37°C overnight

--Karl.brune 22:45, 5 September 2009 (UTC)