Revision as of 13:13, 21 August 2012 by Sato (Talk | contribs)

August 2

Mutation of FT

Inverse PCR
10xBufer2mM dNTPsprimer fwdprimer revtemplatepolymeraseMilliQTotal

94°C 2min, (98°C 10sec, 68°C 4min)x2cycles, 4°C Hold

Dpn1 Digestion
PCR productDpn1
37℃,1h incubate
productMilliQLigaseT4 KinaseTotal

16°C, 1h incubate

competent cellDNA

Cells were stored on ice for 30min.
After 42°C 60sec heat shock, cells were stored on ice for 2min.
Then cells were preincubated at 37°C for 1hr, plated to Kanamycin plate.

August 10

Golden Gate Assembly method This method helps us to constract some genes quickly and we can design the order of constractions. We are trying to construct a gene cycle composed of 6 genes( pSB1A3, lacI, GFP, RFP, GFP, DT). After this experiment is confirmed to work, we will so some experiment to check how the numbers of promorters influences the strength of transcription.

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