Team:KAIT Japan/Protocol

From 2014.igem.org

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<H1>Protocol</H1>
<H1>Protocol</H1>
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: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)
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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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:3) Removed supernatant and  performed 2)operation again, Removed supernatant .
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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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:5) Inverted bacterial pellet by complete fall mixtureing in 200ul Solution Ⅱ resuspension buffer,and confirmed that it became transparent.
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:6) Cooled  for three minutes in ice.
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</p>
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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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</p>
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:8) Cooled  for 3 minutes in ice.
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</p>
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:9) Harvested the DNA by spinning at 10000rpm (4℃) for 10 min.
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</p>
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:10) Gathered only supernatant and moved it in a new microcentrifuge tube.
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</p>
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:11) Added 0.8ul Rnase(10mg/ml) and incubate the solution(37℃,1min)
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</p>
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:12) Added 200ul phenol:chloroform(1:1) and inverted
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</p>
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:13) Harvested  by spinning at 10000rpm (4℃) for 5 min.
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</P>
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:14)  Removed only supernatant and moved it in a new microcentrifuge tube, after that tapped in 200ul chloroform.
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</p>
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:15)  Harvested  by spinning at 10000rpm (4℃) for 1 min.
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</p>
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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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:17)
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Revision as of 03:31, 21 August 2014

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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) 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(50 mM glucose:9g, 25 mM Tris-HCl(pH 8.0):25ml, 10mM EDTA:).

5) Inverted bacterial pellet by complete fall mixtureing in 200ul Solution Ⅱ resuspension buffer,and confirmed that it became transparent.

6) Cooled for three minutes in ice.

7) Inverted bacterial pellet by complete fall mixtureing in 150ul Solution Ⅲ resuspension buffer,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 sodium acetate.(Don't gather underlayer)(The ratio of the 3M sodium acetate and supernatant is made to be 10:1)

17)

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