Team:Tokyo Metropolitan/Notebook/A04

From 2011.igem.org

(Difference between revisions)
(→-Targeting-)
(→-Visualization-)
 
(2 intermediate revisions not shown)
Line 5: Line 5:
= -Targeting- =
= -Targeting- =
 +
 +
-Experiment no.01:transformation for J23100 and P0440
 +
-Methods-
-Methods-
Line 36: Line 39:
*Plasmid solution of J23100 and P0440 are keeping in microfuge tubes and in freezer in Cell-biochemistory lab.
*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.
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 digested 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.

Latest revision as of 10:39, 5 October 2011

-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 digested 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.

Retrieved from "http://2011.igem.org/Team:Tokyo_Metropolitan/Notebook/A04"