Team:Tokyo Metropolitan/Notebook/S12

From 2011.igem.org

(Difference between revisions)
(-Targeting-)
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Totall         20 ul
Totall         20 ul
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-Digestion
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Promoter, suppressed expression system and RFP: Xba1
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-Electrophoresis
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Above samples
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-Digestion
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Promoter: Spe1 and Pst1
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Suppressed expression system and RFP: Xba1 and Pst1
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 +
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-Electrophoresis
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 +
Above samples
 +
 +
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-Gel extraction
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Above samples
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 +
 +
-Electrophoresis to check
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Above samples

Revision as of 13:27, 5 October 2011

-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