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From 2012.igem.org

August 2

Mutation of FT

Inverse PCR

10xBufer	2mM dNTPs	primer fwd	primer rev	template	polymerase	MilliQ	Total
5	5	1.5	1.5	0.5	1	35.5	50

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

Dpn1 Digestion

PCR product	Dpn1
50	2

37℃,1h incubate

Self-ligation

product	MilliQ	Ligase	T4 Kinase	Total
2	7	5	1	15

16°C, 1h incubate

Transformation

competent cell	DNA
20	2

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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