-Targeting-

-Experiment no.01:transformation for J23100 and P0440

-Methods-

Added 10μl distilled water to wells of J23100 and P0440;distribution kit of iGEM. Then pipetted a couple of times, left it for 5min. After, all collected. NovaBlue T1R used as copetentcell, not ECOS-competent cell.

-Protocol-

1. Melt NovaBlue T1R on the ice.

2. Add 2μl plasumid to NovaBlue T1R, and incubate on the ice for 30 min.

3. Heatshock at 42℃, 30 sec.

4. Incubate on the ice for 2 min.

5. Spread 20-250μl on the plate.

6. Incubate at 37℃ for 12-14 hours.

-Results-

E.coli transformed for J23100 and P0440 have been incubating.

-Next experiment-

Check the growth of E.coli transformed for J23100 and P0440.(Shimada 1000-) Make LB medium for preculture.(Shimada & Ichikawa 1500-) Preculture E.coli transformed for J23100 and P0440.(Shimada & Ichikawa 2100-)

• Plasmid solution of J23100 and P0440 are keeping in microfuge tubes and in freezer in Cell-biochemistory lab.

Seven plates of LB medium (Amp 50μg/ml)(didn't use) are keeping in cooling room in Biological macinery room.

-Visualization-

1. Electrophoresis

Run ori/GFP/RFP treated restriction enzyme yesterday.

ori/GFP/RFP

×10buffer：about 3μl

marker: 5μl

Run for about 25min. ori/GFP were cheked bands thus cut gel and refining.

2. Refining

ori: gel 0.093g, QB 279μl

GFP: gel 0.104g, QB 312μl

Left them at 50 deg C for 10 min.

Centrifuged at room tempurature, 13000prm 1 min.

Add 500μl QB.

Centrifuged at room tempurature, 13000prm 1 min.

Add 750μl PE buffer.

Centrifuged at room tempurature, 13000prm 1 min.

Centrifuged at room tempurature, 13000prm 1 min.

Moved calumm to 1.5ml tube.

Add 30μl EB buffer and left it for 1 min.

Centrifuged at room tempurature, 13000prm 1 min.

3. Measurement of concentration of DNA

oriT:10.7 ng/μl

GFP:10.3 ng/μl

Check again to electrophorese because result of absorbance was not good.

4. Electrophoresis

Electrophoresed each 3μl ori/GFP at ×10buffer.

ori made clearly bands and GFP made dimly bands. Thus ligate at ori:GFP = 1:2.

5. Ligation

ori:3μl

GFP:6μl

T4：1μl

×10：2μl

BW:8μl

ori:3μl

T4：1μl

×10：2μl

BW:14μl

Make these two types.

Leave them at 16 deg C for a day. Transform on 5th Aug.