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Team:Bordeaux/NotebookJune23
From 2014.igem.org
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Thermal cycler : <br> | Thermal cycler : <br> | ||
| - | <div style="margin-left: | + | <div style="margin-left:70px ;"> |
3min 95°C<br> | 3min 95°C<br> | ||
| - | + | 25 cycles : 30sec 95°C <br><div style="margin-left:70px ;"> 30sec at all temperatures tested<br> | |
| - | 25 cycles 30sec | + | 3sec 72°C<br></div> |
| - | <div style="margin-left: | + | |
| - | 3sec 72°C<br> | + | |
5min 72°C</div> | 5min 72°C</div> | ||
</li> | </li> | ||
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<tr> | <tr> | ||
<th>DNA</th> | <th>DNA</th> | ||
| - | <td>10µl</td> | + | <td>10µl (*)</td> |
| - | <td>10µl</td> | + | <td>10µl (**)</td> |
</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><br><br> | + | (**) With DNA= 0.56μl Miniprep product + 9,44μl H2O</li><br><br> |
| - | <li>Digestion vector psB6A1 : | + | <li>Digestion vector psB6A1 : <br> |
| + | <img style="margin-left:20px;" src="https://static.igem.org/mediawiki/2014/b/bd/Math1.PNG" alt=math1><br><br> | ||
<table> | <table> | ||
Latest revision as of 00:16, 18 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
25 cycles : 30sec 95°C30sec at all temperatures tested5min 72°C
3sec 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.
