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Team:Bordeaux/NotebookJune23
From 2014.igem.org
(Difference between revisions)
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Test of different hybridation temperature for PCR: 59°C 61°C 63°C 65°C 68°C <br><br> | Test of different hybridation temperature for PCR: 59°C 61°C 63°C 65°C 68°C <br><br> | ||
| - | Mix for 11 tubes | + | Mix for 11 tubes<br> |
<table> | <table> | ||
<tr> | <tr> | ||
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<td>dNTP</td> | <td>dNTP</td> | ||
<td>11µl</td> | <td>11µl</td> | ||
| - | </tr> | + | </tr> |
| - | + | ||
</table><br> | </table><br> | ||
| + | |||
| + | Thermal cycler : <br> | ||
<div style="margin-left:25px ;"> | <div style="margin-left:25px ;"> | ||
| - | |||
| - | |||
3min 95°C<br> | 3min 95°C<br> | ||
30sec 95°C </div><br> | 30sec 95°C </div><br> | ||
| - | 25 cycles | + | 25 cycles 30sec at all temperatures tested<br> |
| - | <div style="margin-left: | + | <div style="margin-left:25px ;"> |
| - | + | ||
3sec 72°C<br> | 3sec 72°C<br> | ||
5min 72°C</div> | 5min 72°C</div> | ||
| - | </ | + | </li> |
| - | <br> | + | <br><br> |
| - | |||
<li>Digestion cloning 1 (psB6A1) miniprep of 10/06/14 and VPGXG-20 (PCR of 10/06/14)<br> | <li>Digestion cloning 1 (psB6A1) miniprep of 10/06/14 and VPGXG-20 (PCR of 10/06/14)<br> | ||
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</tr> | </tr> | ||
</table> | </table> | ||
| - | |||
<br> | <br> | ||
With DNA = 6μl vector + 4μl H2O <br> | With DNA = 6μl vector + 4μl H2O <br> | ||
| - | With DNA= 0.56μl Miniprep product + 9,44μl H2O</li> | + | With DNA= 0.56μl Miniprep product + 9,44μl H2O</li><br><br> |
<li>Digestion vector psB6A1 : | <li>Digestion vector psB6A1 : | ||
Revision as of 23:42, 17 October 2014
23rd June
Molecular biology
- PCR of ELP-40 and ELP-60
=> Dilution primer 100µM => 10µM
Primer forward: 1µl primer + 9µl H2O (Tm=58°C)
Primer reverse: 1µl primer + 9µl H2O (Tm=68°C)
Test of different hybridation temperature for PCR: 59°C 61°C 63°C 65°C 68°C
Mix for 11 tubes
H2O 357,5 µl Buffer 5X 110 µl Primer 27,5 µl dNTP 11µl
Thermal cycler :
3min 95°C
30sec 95°C
25 cycles 30sec at all temperatures tested
3sec 72°C
5min 72°C - Digestion cloning 1 (psB6A1) miniprep of 10/06/14 and VPGXG-20 (PCR of 10/06/14)
Digestion plasmid Digestion insert H2O 7,5µl 7,5µl 10X buffer 4 2µl 2µl XbaI 0,25µl 0,25µL EcoRI 0,25µl 0,25µl DNA 10µl 10µl
With DNA = 6μl vector + 4μl H2O
With DNA= 0.56μl Miniprep product + 9,44μl H2O - Digestion vector psB6A1 :
Digestion plasmid H2O 7,5µl 10X buffer 4 2µl XbaI 0,25µl EcoRI 0,25µl DNA 10µl - Digestion psB1C3 and PCR 40 and 60
OD PCR tube 40= 282,5 ng/μl
OD PCR tube 60= 366,3 ng/μl
Tube 40 = 0,88 DNA +9,12µl H2O
Tube 60 = 2,5 DNA + 7,5 µl H2O
Production
We want to do a preculture test to have a better production.
