Difference between revisions of "Team:TUDelft/Wetlab/Protocols"

Line 36: Line 36:
 
NOTE: mind pipetting errors so prepare at little bit more master mix!. ><br>
 
NOTE: mind pipetting errors so prepare at little bit more master mix!. ><br>
 
For one sample: </li>
 
For one sample: </li>
 +
<br>
 +
<table>
 +
  <tr>
 +
      <th>Component</th>
 +
      <th>Volume (µL)</th>
 +
  </tr>
 +
  <tr>
 +
      <td>GoTaq 5x buffer</td>
 +
      <td>10</td>
 +
  </tr>
 +
  <tr>
 +
      <td>10 mM dNTPs</td>
 +
      <td>1</td>
 +
  </tr>
 +
  <tr>
 +
        <td>Primer VF2 (10µM)</td>
 +
        <td>1</td>
 +
  </tr>
 +
  <tr>
 +
        <td>Primer VR (10µM)</td>
 +
        <td>1</td>
 +
  </tr>
 +
  <tr>
 +
        <td>Sterile milli-Q</td>
 +
        <td>31.8</td>
 +
  </tr>
 +
  <tr>
 +
        <td>Gotaq polymerase (5u/µL)</td>
 +
        <td>0.2</td>
 +
  </tr>
 +
  <tr>
 +
        <td>Total</td>
 +
        <td>45</td>
 +
  </tr>
 +
</table>
 +
* NOTE: Use GoTaq Buffer Green when it is required to run a verification gel afterwards.
 +
 +
<il>Pipette 45 µL of mix into each PCR tube (one tube per colony). </il>
 +
<il>Centrifuge the colony mixture for 5 minutes at 16,000 x g.</il>
 +
<il>Add 5 µL of supernatant of colony mixture to each PCR tube.</il>
 +
<il>Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time): </il>
 +
 +
<br>
 +
<table>
 +
  <tr>
 +
      <th>Step</th>
 +
      <th>Time (s)</th>
 +
      <th>Temperature (°C)</th>
 +
  </tr>
 +
  <tr>
 +
      <td>Initial denaturation</td>
 +
      <td>150</td>
 +
      <td>98</td>
 +
  </tr>
 +
  <tr>
 +
      <td>Denaturation</td>
 +
      <td>60</td>
 +
      <td>94</td>
 +
  </tr>
 +
  <tr>
 +
        <td>Annealing</td>
 +
        <td>60</td>
 +
      <td>60 (depending on primers)</td>
 +
  </tr>
 +
  <tr>
 +
        <td>Extension</td>
 +
        <td>60 sec per kb DNA</td>
 +
      <td>72</td>
 +
  </tr>
 +
  <tr>
 +
        <td>Final extension</td>
 +
        <td>600</td>
 +
      <td>72</td>
 +
  </tr>
 +
  <tr>
 +
        <td>Hold</td>
 +
        <td>∞</td>
 +
      <td>4</td>
 +
  </tr>
 +
</table>
  
 
</ol>
 
</ol>

Revision as of 11:32, 15 September 2018

Wetlab Protocols

Text to write to introduce the protocols


  1. Under sterile conditions, pick a colony and dilute it in 10 µL of milli-Q water.
    NOTE: After you pick the colony, it cannot be used again. It is therefore recommended to make a 'back-up'-plate where you grow the colonies again. This plate should be incubated overnight at 37 °C.
  2. Incubate at 90 °C for 10 min.
    NOTE: Instead of separate 'cooking' of the cells before the PCR, this step can be incorporated in the PCR program. The initial denaturation step at 98 °C should then be prolonged to 5 minutes (300 seconds).
  3. Prepare the GoTaq master mix for all the samples in a single 1.5 mL tube
    NOTE: mind pipetting errors so prepare at little bit more master mix!. >
    For one sample:

    4. Component Volume (µL)
    GoTaq 5x buffer 10
    10 mM dNTPs 1
    Primer VF2 (10µM) 1
    Primer VR (10µM) 1
    Sterile milli-Q 31.8
    Gotaq polymerase (5u/µL) 0.2
    Total 45
    * NOTE: Use GoTaq Buffer Green when it is required to run a verification gel afterwards. Pipette 45 µL of mix into each PCR tube (one tube per colony). Centrifuge the colony mixture for 5 minutes at 16,000 x g. Add 5 µL of supernatant of colony mixture to each PCR tube. Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time):
    Step Time (s) Temperature (°C)
    Initial denaturation 150 98
    Denaturation 60 94
    Annealing 60 60 (depending on primers)
    Extension 60 sec per kb DNA 72
    Final extension 600 72
    Hold ∞ 4