Team:Biwako Nagahama/general protocol

From 2013.igem.org

(Difference between revisions)
(→Genaral protocol)
(→Genaral protocol)
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     <div class="top-sentence">
     <div class="top-sentence">
== <h2>Genaral protocol</h2> ==
== <h2>Genaral protocol</h2> ==
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<h3><Colony PCR>(check Trance formation)</h3>
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<h3>[https://www.bioline.com/h_prod_detail.asp?itemid=219 BIOTAQ™ DNA Polymerase]PCR</h3>
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<p>Take a single colony from the plate and then mix it with 30μL of de-ionized water(dH2O)</p>
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<div style="background: white; border: 1px solid black; padding: 1em;margin: 0 3em;">
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<p>↓</p>
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<table border="1">
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<tr><th>aaa</th><th>アルバム</th></tr>
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<tr><td>1993</td><td>Janet</td></tr>
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<tr><td>1997</td><td>Control</td></tr>
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<tr><td>1997</td><td>The belbet robe</td></tr>
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<tr><td>2001</td><td>All for you</td></tr>
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</table>
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</div>
<p>dH2O・・・12.7μL</p>
<p>dH2O・・・12.7μL</p>
<p>10×NH4 buffer・・・2.5μL</p>
<p>10×NH4 buffer・・・2.5μL</p>
<p>50mM MgCl2・・・0.75μL</p>
<p>50mM MgCl2・・・0.75μL</p>
<p>25mM dNTPs・・・2μL</p>
<p>25mM dNTPs・・・2μL</p>
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<p>10μM [http://parts.igem.org/Part:BBa_G1004 ※3] BBa_G1004・・・1μL</p>
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<p>10μM F Primer・・・1μL</p>
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<p>10μM [http://parts.igem.org/Part:BBa_G1005 ※4] BBa_G1005・・・1μL</p>
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<p>10μM R Primer・・・1μL</p>
<p>BIO Taq(5U/μL)・・・0.05μL</p>
<p>BIO Taq(5U/μL)・・・0.05μL</p>
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<p>Template(dH2O mixed colony)・・・5μL</p>
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<p>Template(<500ng)・・・5μL</p>
<p>Total・・・25μL</p>
<p>Total・・・25μL</p>
<p>[https://www.bioline.com/h_prod_detail.asp?itemid=219 BIOTAQ™ DNA Polymerase]</p>
<p>[https://www.bioline.com/h_prod_detail.asp?itemid=219 BIOTAQ™ DNA Polymerase]</p>

Revision as of 18:13, 27 September 2013

Contents

Genaral protocol

BIOTAQ™ DNA PolymerasePCR

aaaアルバム
1993Janet
1997Control
1997The belbet robe
2001All for you

dH2O・・・12.7μL

10×NH4 buffer・・・2.5μL

50mM MgCl2・・・0.75μL

25mM dNTPs・・・2μL

10μM F Primer・・・1μL

10μM R Primer・・・1μL

BIO Taq(5U/μL)・・・0.05μL

Template(<500ng)・・・5μL

Total・・・25μL

BIOTAQ™ DNA Polymerase

↓

95℃　30sec

↓

95℃　10sec

55℃ 20sec 30cycles

72℃ 2min

↓

72℃　2min

↓

10℃　∞

Distribution kit

↓With a pipette tip, punch a hole in the foil

↓Add 10μL of dH2O,and pipetting

↓Put 5min

↓Pipette 1uL of the resuspended DNA Transformation into your desired 100μL of competent cells

↓Hold on ice for 20min

↓Heat shock at 42℃ for 30sec

↓quickly

↓On ice for 2min

↓Add 900μL of SOCborth

↓Hold at 37℃ for 30min

↓Plating 100μL of DNA Transformation

↓Centrifuge for 1 min(13,000rpm)

↓Waste supernatant for 800μL, and pipetting

↓Plating all

↓Incubate at 37℃ (over night)

Phenol-chloroform extraction

Add Phenol-chloroform where is equivalent to Exo Star process sample

↓Centrifuge 4℃ 13,000rpm 5min

Only supernatant was taken

EtOH crystalization

↓Add 1μL 20mg/mL Glycogen

↓Mix

↓Add 1/10 volume 3M CH3COONa(pH5.2)

↓Add 2.5 times volume 99.5% EtOH

↓Vortex

↓Centrifuge 4℃ 13,000rpm 20min

↓Waste supernatant

↓Add 500μL 70% EtOH

↓Mix

↓Centrifuge for 10s in a table-top microcentrifuge

↓Waste supernatant

↓65℃ Dry up

↓Add 11μL TE buffer

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