Team:KAIT Japan/Protocol

From 2014.igem.org

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<H1>Protocol</H1>
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<H1><font size="7">Protocol</font></H1>
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<font size="5">1:miniprep</font>
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:2) Aliquot 1ml culture into a 1.5 ml microcentrifuge tube,and Made it spin at 10000rpm (4℃) for 1 min to harvest the bacteria.
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:2) Aliquot 1ml culture into a 1.5 ml microcentrifuge tube,and Made it spin at 10000rpm (4℃) for 1 min to harvest the bacteria.
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:4) Resuspended bacterial pellet by complete vortexing in 100ul SolutionⅠ(50 mM glucose:9g, 25 mM Tris-HCl(pH 8.0):25ml, 10mM EDTA:).
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:4) Resuspended bacterial pellet by complete vortexing in 100ul SolutionⅠ{D-glucose:9g(50mM),1M Tris-HCl(pH 8.0):25ml(25mM),0.5M EDTA:20ml(10mM),H<sub>2</sub>O:955ml /1L}.
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:5) Inverted bacterial pellet by complete fall mixtureing in 200ul Solution Ⅱ resuspension buffer,and confirmed that it became transparent.
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:5) Inverted bacterial pellet by complete fall mixtureing in 200ul SolutionⅡ{NaOH:8g,SDS:10g[1%(w/v)],H<sub>2</sub>O:960ml /1L},and confirmed that it became transparent.
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:7) Inverted bacterial pellet by complete fall mixtureing in 150ul Solution Ⅲ resuspension buffer,and confirmed that it became Cloudiness.
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:7) Inverted bacterial pellet by complete fall mixtureing in 150ul SolutionⅢ{CH<sub>3</sub>COOH:294.5g(3M),CH<sub>3</sub>COOH:120ml(2M),H<sub>2</sub>O:diluting in measuring cylinder to 1L total},and confirmed that it became Cloudiness.
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:16) Moved its supernatant to a new microcentrifuge tube and add 15ul 3M sodium acetate.(Don't gather underlayer)(The ratio of the 3M sodium acetate and supernatant is made to be 10:1)
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:16) Moved its supernatant to a new microcentrifuge tube and add 15ul 3M CH<sub>3</sub>COONa.(Don't gather underlayer)(The ratio of the 3M sodium acetate and supernatant is made to be 10:1)
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:17)  
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:17) Add 400ul 100%CH<sub>3</sub>CH<sub>2</sub>OH and made it stirred well.
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:18) Harvested  by spinning at 10000rpm (4℃) for 20 min.
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:19) Removed only supernatant and added 400ul 70%CH<sub>3</sub>CH<sub>2</sub>OH(pour a liquid from the other side for white thing not to drain a white.[white thing is plasmid])
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:20) Harvested  by spinning at 10000rpm (4℃) for 20 min.
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:21) Removed only supernatant and opened the cover of the tube for 10min to dry CH<sub>3</sub>CH<sub>2</sub>OH.
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:22) Add 50ul TE to dissolve DNA
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:23) We stored low temperature
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Revision as of 06:45, 21 August 2014

ロゴ2.png

KAIT Japan2013 Kanako.png

KAIT Japan 2014 iGEMRogo.png
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Home

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Team

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Project

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Parts

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Protocol

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Notebook

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Results

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Safety

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Human Practice

Protocol

1:miniprep

1) We cultured bacterial strain with the LB medium which I added ampicillin to so that density becomes 100ug/ml overnight.(We made a nutrient medium of around 5 ml in 50 ml falcons)(Against 5 ml of nutrient mediums, Amp used 5ul)

2) Aliquot 1ml culture into a 1.5 ml microcentrifuge tube,and Made it spin at 10000rpm (4℃) for 1 min to harvest the bacteria.

3) Removed supernatant and performed 2)operation again, Removed supernatant .

4) Resuspended bacterial pellet by complete vortexing in 100ul SolutionⅠ{D-glucose:9g(50mM),1M Tris-HCl(pH 8.0):25ml(25mM),0.5M EDTA:20ml(10mM),H2O:955ml /1L}.

5) Inverted bacterial pellet by complete fall mixtureing in 200ul SolutionⅡ{NaOH:8g,SDS:10g[1%(w/v)],H2O:960ml /1L},and confirmed that it became transparent.

6) Cooled for three minutes in ice.

7) Inverted bacterial pellet by complete fall mixtureing in 150ul SolutionⅢ{CH3COOH:294.5g(3M),CH3COOH:120ml(2M),H2O:diluting in measuring cylinder to 1L total},and confirmed that it became Cloudiness.

8) Cooled for 3 minutes in ice.

9) Harvested the DNA by spinning at 10000rpm (4℃) for 10 min.

10) Gathered only supernatant and moved it in a new microcentrifuge tube.

11) Added 0.8ul Rnase(10mg/ml) and incubate the solution(37℃,1min)

12) Added 200ul phenol:chloroform(1:1) and inverted

13) Harvested by spinning at 10000rpm (4℃) for 5 min.

14) Removed only supernatant and moved it in a new microcentrifuge tube, after that tapped in 200ul chloroform.

15) Harvested by spinning at 10000rpm (4℃) for 1 min.

16) Moved its supernatant to a new microcentrifuge tube and add 15ul 3M CH3COONa.(Don't gather underlayer)(The ratio of the 3M sodium acetate and supernatant is made to be 10:1)

17) Add 400ul 100%CH3CH2OH and made it stirred well.

18) Harvested by spinning at 10000rpm (4℃) for 20 min.

19) Removed only supernatant and added 400ul 70%CH3CH2OH(pour a liquid from the other side for white thing not to drain a white.[white thing is plasmid])

20) Harvested by spinning at 10000rpm (4℃) for 20 min.

21) Removed only supernatant and opened the cover of the tube for 10min to dry CH3CH2OH.

22) Add 50ul TE to dissolve DNA

23) We stored low temperature

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