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Team:Biwako Nagahama/general protocol
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| - | <tr><th> | + | <tr><th>dH2O</th><th>12.7μL</th></tr> |
| - | <tr><td> | + | <tr><td>10×NH4 buffer</td><td>2.5μL</td></tr> |
| - | <tr><td> | + | <tr><td>50mM MgCl2</td><td>0.75μL</td></tr> |
| - | <tr><td> | + | <tr><td>25mM dNTPs</td><td>2μL</td></tr> |
| - | <tr><td> | + | <tr><td>10μM F Primer</td><td>1μL</td></tr> |
| + | <tr><td>10μM R Primer</td><td>1μL</td></tr> | ||
| + | <tr><td>BIO Taq(5U/μL)</td><td>0.05μL</td></tr> | ||
| + | <tr><td>Template(<500ng)</td><td>5μL</td></tr> | ||
| + | <tr><td>Total</td><td>25μL</td></tr> | ||
</table> | </table> | ||
</div> | </div> | ||
Revision as of 18:20, 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 |
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℃ ∞
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
