Team:Biwako Nagahama/general protocol

From 2013.igem.org

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== <h2>Genaral protocol</h2> ==
== <h2>Genaral protocol</h2> ==
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<h3>[https://www.bioline.com/h_prod_detail.asp?itemid=219 BIOTAQ™ DNA Polymerase]PCR</h3>
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----
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<div style>
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<table border="1">
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<tr><td>dH2O</td><td>12.7μL</td></tr>
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<tr><td>10×NH4 buffer</td><td>2.5μL</td></tr>
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<tr><td>50mM MgCl2</td><td>0.75μL</td></tr>
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<tr><td>25mM dNTPs</td><td>2μL</td></tr>
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<tr><td>10μM F Primer</td><td>1μL</td></tr>
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<tr><td>10μM R Primer</td><td>1μL</td></tr>
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<tr><td>BIO Taq(5U/μL)</td><td>0.05μL</td></tr>
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<tr><td>Template(<500ng)</td><td>5μL</td></tr>
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<tr><td>Total</td><td>25μL</td></tr>
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</table>
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</div>
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----
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<p>95℃　30sec</p>
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<p>↓</p>
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<p>95℃　10sec</p>   
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<p>55℃  20sec&nbsp;&nbsp;&nbsp;&nbsp;30cycles</p>
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<p>72℃  2min</p>
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<p>↓</p>
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<p>72℃　2min</p>
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<p>↓</p>
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<p>10℃　∞</p>
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----
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<h3>[http://www.toyobo-global.com/seihin/xr/lifescience/products/pcr_002.html TOYOBO KOD Fx]PCR</h3>
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----
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<div style>
 +
<table border="1">
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<tr><td>2×KODFx buffer</td><td>25μL</td></tr>
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<tr><td>dNTPs[2mM]</td><td>10μL</td></tr>
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<tr><td>10μM F Primer</td><td>1.5μL</td></tr>
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<tr><td>10μM R Primer</td><td>1.5μL</td></tr>
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<tr><td>KOD Fx[1.0U/μL]</td><td>1.0μL</td></tr>
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<tr><td>MilliQ H2O</td><td>10.0μL</td></tr>
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<tr><td>Template(<500ng)</td><td>1.0μL</td></tr>
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<tr><td>Total</td><td>50μL</td></tr>
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</table>
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</div>
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----
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<p>94℃　2min</p>
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<p>↓</p>
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<p>98℃　10sec</p>   
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<p>58℃  30sec&nbsp;&nbsp;&nbsp;&nbsp;30cycles</p>
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<p>68℃  2min30sec</p>
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<p>↓</p>
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<p>10℃　∞</p>
 +
----
<h3>Distribution kit</h3>
<h3>Distribution kit</h3>
----
----
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<p>Only supernatant was taken</p>
<p>Only supernatant was taken</p>
----
----
-
<h3>EtOH crystalization</h3>
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<h3>EtOH precipitation</h3>
----
----
<p>↓Add 1μL 20mg/mL Glycogen</p>
<p>↓Add 1μL 20mg/mL Glycogen</p>
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<p>↓65℃ Dry up</p>
<p>↓65℃ Dry up</p>
<p>↓Add 11μL TE buffer</p>
<p>↓Add 11μL TE buffer</p>
 +
----
 +
<h3>Miniprep</h3>
 +
----
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<p>1.5mL Culture fluid→New 1.5mL tube</p>
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<p>↓Centrifuge 4℃ 13,000rpm 1min</p>
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<p>Waste supernatant</p>
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<p>↓</p>
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<p>Add 200μl SolutionⅠ</p>
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<p>↓vortex</p>
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<p>Add 200μl SolutionⅡ</p>
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<p>↓Mix(softy)</p>
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<p>Add 200μl SolutionⅢ</p>
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<p>↓Mix(softy)</p>
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<p>Check to become cloudy</p>
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<p>↓</p>
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<p>On ice 5min</p>
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<p>↓Centrifuge 4℃ 13,000rpm 10min</p>
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<p>supernatant→New 1.5mL tube</p>
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<p>↓</p>
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Add 500μL Isopropyl alchol
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<p>↓vortex</p>
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<p>↓Centrifuge 4℃ 13,000rpm 20min</p>
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<p>Waste supernatant</p>
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<p>↓</p>
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<p>Add 500μL 70% EtOH</p>
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<p>↓Mix</p>
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<p>↓Flash</p>
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<p>Waste supernatant</p>
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<p>↓</p>
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<p>65℃ Dry up</p>
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<p>↓</p>
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<p>Add 50μL RNase in TE buffer</p>
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<p>↓Mix</p>
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<p>37℃ 20min</p>
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<p></p>
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By. Syohei Takeshita
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Latest revision as of 23:01, 27 September 2013

Contents

Genaral protocol

BIOTAQ™ DNA PolymerasePCR

dH2O12.7μL
10×NH4 buffer2.5μL
50mM MgCl20.75μL
25mM dNTPs2μL
10μM F Primer1μL
10μM R Primer1μL
BIO Taq(5U/μL)0.05μL
Template(<500ng)5μL
Total25μL

95℃　30sec

↓

95℃　10sec

55℃ 20sec    30cycles

72℃ 2min

↓

72℃　2min

↓

10℃　∞

[http://www.toyobo-global.com/seihin/xr/lifescience/products/pcr_002.html TOYOBO KOD Fx]PCR

2×KODFx buffer25μL
dNTPs[2mM]10μL
10μM F Primer1.5μL
10μM R Primer1.5μL
KOD Fx[1.0U/μL]1.0μL
MilliQ H2O10.0μL
Template(<500ng)1.0μL
Total50μL

94℃　2min

↓

98℃　10sec

58℃ 30sec    30cycles

68℃ 2min30sec

↓

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 precipitation

↓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

Miniprep

1.5mL Culture fluid→New 1.5mL tube

↓Centrifuge 4℃ 13,000rpm 1min

Waste supernatant

↓

Add 200μl SolutionⅠ

↓vortex

Add 200μl SolutionⅡ

↓Mix(softy)

Add 200μl SolutionⅢ

↓Mix(softy)

Check to become cloudy

↓

On ice 5min

↓Centrifuge 4℃ 13,000rpm 10min

supernatant→New 1.5mL tube

↓

Add 500μL Isopropyl alchol

↓vortex

↓Centrifuge 4℃ 13,000rpm 20min

Waste supernatant

↓

Add 500μL 70% EtOH

↓Mix

↓Flash

Waste supernatant

↓

65℃ Dry up

↓

Add 50μL RNase in TE buffer

↓Mix

37℃ 20min

By. Syohei Takeshita

Retrieved from "http://2013.igem.org/Team:Biwako_Nagahama/general_protocol"