Difference between revisions of "Team:Paris Bettencourt/Notebook/VitaminB2"

Line 34: Line 34:
 
     <td>98</td>
 
     <td>98</td>
 
     <td>melting</td>
 
     <td>melting</td>
 +
  </tr>
 +
 +
  <tr>
 +
    <td> </td>
 
   </tr>
 
   </tr>
  
Line 68: Line 72:
 
   </tr>
 
   </tr>
 
</table>
 
</table>
−
<li>30sec at 98°C</br>
 
−
</br>12 cycles</br>
 
−
<li>      10sec at 98°C</br>
 
−
<li>      30sec at 52°C</br>
 
−
<li>    1min at 72°C</br>
 
−
</br>
 
−
<li>10min at 72°C</br>
 
−
until opening at 12°C</br>
 
−
</br>
 

Revision as of 15:13, 10 August 2015

13/07
  • Received gBlocks RibA, RibD, RibE, RibT25 and RibT48 and amplification oligos from IDT.
    • Dilution in water and PCR amplification, using the following protocol:
    • 1 μL gBlock (0.1 to 1ng)
    • 1 μL forward primer (10 μM)
    • 1 μL reverse primer (10 μM)
    • 22 μL DNAse/RNAse free water
    • 25 μL LifeTech MasterMix (2X)

    RibA + o15.001 (GCGCCCGAAGACTTATGCAG) + o15.002(GGCCCCGCGCATATGAAG)
    RibD + o15.003(CGCTATAGAAGACTTGAGAAGATCTG) + o15.004(GCGCGGCACCACATATGAAG)
    RibE + o15.005(CGGCTATAGAAGACTTGCGC) + o15.006(CGCGCCGGCATATGAAGA)
    RibT25 + o15.007(CCGCGTATAGAAGACTGCTAGA) + o15.008(CAGCAGCATATGAAGACAACCC)
    RibT48 + o15.009(GCGGTATAGAAGACTGCTAGAGA) + o15.010(CAGCAGCATATGAAGACAACCC)

    For each PCR reaction, a negative control without matrix DNA was prepared.

    time (min) temperature (°C) function
    3:00 98 melting
    0:30 98 melting
    0:30 52 annealing
    1:00 72 extension
    10:00 72 extension
    forever 12 storage