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Team:Biwako Nagahama/general protocol
From 2013.igem.org
(→Genaral protocol) |
(→Genaral protocol) |
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| Line 25: | Line 25: | ||
<p>↓</p> | <p>↓</p> | ||
<p>95℃ 10sec</p> | <p>95℃ 10sec</p> | ||
| - | <p>55℃ 20sec | + | <p>55℃ 20sec 30cycles</p> |
<p>72℃ 2min</p> | <p>72℃ 2min</p> | ||
<p>↓</p> | <p>↓</p> | ||
| Line 33: | Line 33: | ||
---- | ---- | ||
<h3>[http://www.toyobo-global.com/seihin/xr/lifescience/products/pcr_002.html TOYOBO KOD Fx]PCR</h3> | <h3>[http://www.toyobo-global.com/seihin/xr/lifescience/products/pcr_002.html TOYOBO KOD Fx]PCR</h3> | ||
| + | ---- | ||
| + | <div style> | ||
| + | <table border="1"> | ||
| + | <tr><td>2×KODFx buffer</td><td>25μL</td></tr> | ||
| + | <tr><td>dNTPs[2mM]</td><td>10μL</td></tr> | ||
| + | <tr><td>10μM F Primer</td><td>1.5μL</td></tr> | ||
| + | <tr><td>10μM R Primer</td><td>1.5μL</td></tr> | ||
| + | <tr><td>KOD Fx[1.0U/μL]</td><td>1.0μL</td></tr> | ||
| + | <tr><td>MilliQ H2O</td><td>10.0μL</td></tr> | ||
| + | <tr><td>Template(<500ng)</td><td>1.0μL</td></tr> | ||
| + | <tr><td>Total</td><td>50μL</td></tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | ---- | ||
| + | <p>94℃ 2min</p> | ||
| + | <p>↓</p> | ||
| + | <p>98℃ 10sec</p> | ||
| + | <p>58℃ 30sec 30cycles</p> | ||
| + | <p>68℃ 2min30sec</p> | ||
| + | <p>↓</p> | ||
| + | <p>10℃ ∞</p> | ||
---- | ---- | ||
<h3>Distribution kit</h3> | <h3>Distribution kit</h3> | ||
| Line 58: | Line 79: | ||
<p>Only supernatant was taken</p> | <p>Only supernatant was taken</p> | ||
---- | ---- | ||
| - | <h3>EtOH | + | <h3>EtOH precipitation</h3> |
---- | ---- | ||
<p>↓Add 1μL 20mg/mL Glycogen</p> | <p>↓Add 1μL 20mg/mL Glycogen</p> | ||
| Line 73: | Line 94: | ||
<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> | ||
| + | ---- | ||
| + | <p>1.5mL Culture fluid→New 1.5mL tube</p> | ||
| + | <p>↓Centrifuge 4℃ 13,000rpm 1min</p> | ||
| + | <p>Waste supernatant</p> | ||
| + | <p>↓</p> | ||
| + | <p>Add 200μl SolutionⅠ</p> | ||
| + | <p>↓vortex</p> | ||
| + | <p>Add 200μl SolutionⅡ</p> | ||
| + | <p>↓Mix(softy)</p> | ||
| + | <p>Add 200μl SolutionⅢ</p> | ||
| + | <p>↓Mix(softy)</p> | ||
| + | <p>Check to become cloudy</p> | ||
| + | <p>↓</p> | ||
| + | <p>On ice 5min</p> | ||
| + | <p>↓Centrifuge 4℃ 13,000rpm 10min</p> | ||
| + | <p>supernatant→New 1.5mL tube</p> | ||
| + | <p>↓</p> | ||
| + | Add 500μL Isopropyl alchol | ||
| + | <p>↓vortex</p> | ||
| + | <p>↓Centrifuge 4℃ 13,000rpm 20min</p> | ||
| + | <p>Waste supernatant</p> | ||
| + | <p>↓</p> | ||
| + | <p>Add 500μL 70% EtOH</p> | ||
| + | <p>↓Mix</p> | ||
| + | <p>↓Flash</p> | ||
| + | <p>Waste supernatant</p> | ||
| + | <p>↓</p> | ||
| + | <p>65℃ Dry up</p> | ||
| + | <p>↓</p> | ||
| + | <p>Add 50μL RNase in TE buffer</p> | ||
| + | <p>↓Mix</p> | ||
| + | <p>37℃ 20min</p> | ||
| + | <p></p> | ||
| + | By. Syohei Takeshita | ||
<div class="spacer"></div> | <div class="spacer"></div> | ||
</div> | </div> | ||
<div class="spacer"></div> | <div class="spacer"></div> | ||
</div> | </div> | ||
Latest revision as of 23:01, 27 September 2013
Contents |
Genaral protocol
BIOTAQ™ DNA PolymerasePCR
| 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 |
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 buffer | 25μL |
| dNTPs[2mM] | 10μL |
| 10μM F Primer | 1.5μL |
| 10μM R Primer | 1.5μL |
| KOD Fx[1.0U/μL] | 1.0μL |
| MilliQ H2O | 10.0μL |
| Template(<500ng) | 1.0μL |
| Total | 50μL |
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
