-Speeding Up-

-Treat restriction enzyme

HNS(wt) -> X,S

I120260 -> Bln I

for two hours.

-DNA refining

Phenol/Chloroform preparation and ethanol precipitation

-Measurement of DNA concentration

0.04 ug/ml

-Ligation

HNS(wt):I20260 (expect GFP) = 3:1

-Transformation Transformed DAN solution.

-Visualization-

-Plasmid extraction

-Treat restriction enzyme (only S)

-Electrophoresis

Results

Cleared plasmid extraction but retest to check for the protocol.

Checked plates

Checked plates transformed after ligate one day ago. Checked colony on all plates J23100:K317037 = 1:4,1:8 and 1:0.

Treated restriction enzyme

Treated P0440 restriction enzyme with xba and pst and incubated at 37 deg C for 6 hours.

Colony PCR

Did colony PCR for 32 colony (16 colony for J23100:K317037=1:4, 14 colony for J23100:K317037=1:8 and 2 colony for only J23100).

Master plates have been incubating.

Electrophoresis

Checked results for colony PCR and treated restriction enzyme by electrophoresis. Showed two bands with colony PCR. Did not show bands with P0440 treated restriction enzyme.

Pre-cluture

Pre-clutured six tubes : three P0440 (named Mr.P, P-chan and P-ko) and colony of master plates (10,16 and 26). Liquid midum contained ampicillin. Colony of master plates used showing a band by electrophoresis. Incubated at 37 deg C after pre-cluture.

-Electrophoresis

-Gel refining

-Electrophoresis

-Digestion

Details

-Electrophoresis

Checking for OriTr + GFP (1,2) and OriTr (3,4)

Marker was 5 ul Gene ladder wide.

Chceked four binds.

-Gel refining

c.f. 4th Aug.

Refined with 1 and 2, 3 and 4 in tubes.

-Electrophoresis

Checked for complete to gel refine.

Run 3 ul + 3 ul Loading buffer.

Marker was 5 ul Gen ladder wide.

Did not show bands.

• Used sample without ethanol when gel refining.

-Digestion

Did redoing because could not gel refinging.

-Targeting-

Checked plates

Checked plates transformed after ligate one day ago. Checked colony on all plates J23100:K317037 = 1:4,1:8 and 1:0.

Digestion

Digested P0440 with xba and pst and incubated at 37 deg C for 6 hours.

Colony PCR

Did colony PCR for 32 colony (16 colony for J23100:K317037=1:4, 14 colony for J23100:K317037=1:8 and 2 colony for only J23100).

Master plates have been incubating.

Electrophoresis

Checked results for colony PCR and treated restriction enzyme by electrophoresis. Showed two bands with colony PCR. Did not show bands with P0440 treated restriction enzyme.

Pre-cluture

Pre-clutured six tubes : three P0440 (named Mr.P, P-chan and P-ko) and colony of master plates (10,16 and 26). Liquid midum contained ampicillin. Colony of master plates used showing a band by electrophoresis. Incubated at 37 deg C after pre-cluture.