-Targeting-

-Electrophoresis Checked to treat restriction enzyme for J04450 and J23100-P0440. Cut only J23100-P0440.

-Plasmid extraction and DNA refining

Extracted plasmids and refined DNA for J23100-K317037, J23100-P0440, K283030 and J22005.

Results (concentration = ng/ul)

J2310-K317037 (Jack) was 60

J23100-P0440 (Jamper) was 120

K283030 (Kasuga) was 155

J22005 (Jin) was 183

-Treat restriction enzyme

Treated restriction enzyme with E and P for J04450. After, incubated at 37 deg C for 5 hours.

-Electrophoresis

Checked to treat restriction enzyme for J04450. Results was a failure.

-Treat restriction enzyme

Jack -> S and P

Jamper -> E and P

Kasuga -> X and P

Jin -> Nhe

had been incubated at 37 deg C after treat restriction enzyme with preceding enzymes.

-Visualization-

-Digestion

OriTr-GFP

EcoRⅠ 1 ul

SpeⅠ　 1 ul

plasmid 4 ul

10×H 1.75 ul

DW 9.75 ul

Totall 17.5 ul

-OriTr

EcoRⅠ 1 ul

XbaⅠ　 1 ul

plasmid 4 ul

10×H 1.75 ul

DW 9.75 ul

Totall 17.5 ul

-Electrophoresis

According to protocol

-Gel extraction

Used QIAGEN's kits

-Electrophoresis to check

Run 3 ul.

Checked bands.

-Ligation

OriTr 　　　　　　　 2 ul

OriTr-GFP　　　　　　6 ul　

T4 Ligase　　　　　　1 ul

10×Ligation buffur　2 ul

DW　　　　　　　　　　9 ul

Totall　　　　　　　　　20 ul

-Digestion

Promoter, suppressed expression system and RFP: Xba1

-Electrophoresis

Above samples

-Digestion

Promoter: Spe1 and Pst1

Suppressed expression system and RFP: Xba1 and Pst1

-Electrophoresis

Above samples

-Gel extraction

Above samples

-Electrophoresis to check

Above samples