-Targeting-

-Plasmid extraction and DNA refining

Extracted plasmid and reined DNA of J23100, P0140 and John-K-Kennedy precultured one day ago.

Results (ng/ul)

J21300 54

P0140 55

John-K-Kennedy

I. 47

II. 46

-Digestion

Digested with P and S for J23100 and John-F-Kennedy, X and P for P0140. Then incubated at 37 deg C.

-Electrophoresis

Checked for treat restriction enzyme

Results

Succeeded to cut J23100, John-F-Kennedy and P0140.

-Gel extraction

Extracted from gel for J23100 and P0140.

Results (ng/ul)

J23100 5

P0140 4

-Ligation and Transformation

Ligated J23100 and P0140 then transformed. Incubated at 37 deg C.

-Visualization-

-Colony PCR for pSB1C3+oriTr-GFP

Did colony PCR for five colony transformed on 20th Sep.

-Electrophoresis for oriTr4,oriTr-GFP 0A,oriTr-GFP 0B

Used plasmid solution, but bands showed thin. Thus restarted from plasmid extraction.

-Pre-culture for oriTr,oriTr-GFP

-Pre-culture for oriTr-GFP-oriTr

-Colony PCR

Samples which transformed one day ago

-Digestion

Promoter: Spe and Pst

Suppressed expression system: Xba and Pst

Over night