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<div class="inner" style="display:none;"> | <div class="inner" style="display:none;"> | ||
<br> <br> | <br> <br> | ||
− | <b> | + | |
− | <b> | + | <b>1. What is the PCR ? </b><br> |
+ | <b>p</b>olymerase <b>c</b>hain <b>r</b>eaction<br> | ||
Method to make multiple copies of a | Method to make multiple copies of a | ||
the specific DNA-sequence <br> | the specific DNA-sequence <br> | ||
− | |||
− | |||
− | |||
− | |||
− | <b>Reaction Setup</b> | + | <b>2. Reaction Setup</b> |
<div style="text-align: justify; margin-left:20px"> | <div style="text-align: justify; margin-left:20px"> | ||
− | 1. | + | 1. Assemble all reaction components on ice, work on ice while assembling <br> |
− | 2. | + | 2. Preheat the thermocycler to the denaturation temperature( 98 °C) <br> |
− | 3. | + | 3. Prior to use all components should be mixed <br> |
− | 4. | + | 4. Work quickly when transferring the reactions to a thermocycler </div> |
<b>Steps </b> | <b>Steps </b> | ||
<div style="text-align: justify; margin-left:40px"> | <div style="text-align: justify; margin-left:40px"> | ||
− | 1. Assemble all components on ice for the reaction : | + | 1. Assemble all components on ice for the reaction :<br> |
<style type="text/css"> | <style type="text/css"> | ||
.tg {border-collapse:collapse;border-spacing:0;} | .tg {border-collapse:collapse;border-spacing:0;} | ||
Line 603: | Line 600: | ||
<table class="tg"> | <table class="tg"> | ||
<tr> | <tr> | ||
− | <th class="tg-yw4l">Component</th> | + | <th class="tg-yw4l" align="center"><b>Component</b></th> |
− | <th class="tg-yw4l">25 μl Reaction</th> | + | <th class="tg-yw4l" align="center"><b>25 μl Reaction</b></th> |
− | <th class="tg-yw4l"> | + | <th class="tg-yw4l" align="center"><b> |
− | 50 μl Reaction</th> | + | 50 μl Reaction</b></th> |
− | <th class="tg-yw4l">Final Concentration</th> | + | <th class="tg-yw4l" align="center"><b>Final Concentration</b></th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td class="tg-yw4l">Q5 | + | <td class="tg-yw4l" align="center">Q5 |
High-Fidelity 2X Master Mix</td> | High-Fidelity 2X Master Mix</td> | ||
− | <td class="tg-yw4l">12.5 μl</td> | + | <td class="tg-yw4l" align="center">12.5 μl</td> |
− | <td class="tg-yw4l">25 μl</td> | + | <td class="tg-yw4l" align="center">25 μl</td> |
− | <td class="tg-yw4l">1X</td> | + | <td class="tg-yw4l" align="center">1X</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td class="tg-yw4l">10 μM Forward Primer</td> | + | <td class="tg-yw4l" align="center">10 μM Forward Primer</td> |
− | <td class="tg-yw4l">1.25 μl</td> | + | <td class="tg-yw4l" align="center">1.25 μl</td> |
− | <td class="tg-yw4l"> | + | <td class="tg-yw4l" align="center"> |
2.5 μl</td> | 2.5 μl</td> | ||
− | <td class="tg-yw4l">0.5 μM</td> | + | <td class="tg-yw4l"align="center">0.5 μM</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td class="tg-yw4l"> | + | <td class="tg-yw4l"align="center"> |
10 μM Reverse Primer</td> | 10 μM Reverse Primer</td> | ||
− | <td class="tg-yw4l">1.25 μl</td> | + | <td class="tg-yw4l"align="center">1.25 μl</td> |
− | <td class="tg-yw4l">2.5 μl</td> | + | <td class="tg-yw4l"align="center">2.5 μl</td> |
− | <td class="tg-yw4l">0.5 μM</td> | + | <td class="tg-yw4l"align="center">0.5 μM</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td class="tg-yw4l"> | + | <td class="tg-yw4l"align="center"> |
Template DNA</td> | Template DNA</td> | ||
− | <td class="tg-yw4l">variable</td> | + | <td class="tg-yw4l"align="center">variable</td> |
− | <td class="tg-yw4l">variable</td> | + | <td class="tg-yw4l"align="center">variable</td> |
− | <td class="tg-yw4l">< 1,000 ng</td> | + | <td class="tg-yw4l"align="center">< 1,000 ng</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td class="tg-yw4l"> | + | <td class="tg-yw4l"align="center"> |
Nuclease-Free Water</td> | Nuclease-Free Water</td> | ||
− | <td class="tg-yw4l">to 25 μl</td> | + | <td class="tg-yw4l"align="center">to 25 μl</td> |
− | <td class="tg-yw4l">to 50 μl</td> | + | <td class="tg-yw4l"align="center">to 50 μl</td> |
− | <td class="tg-yw4l"></td> | + | <td class="tg-yw4l"align="center"></td> |
</tr> | </tr> | ||
</table> | </table> | ||
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NEB #M0212 | NEB #M0212 | ||
) | ) | ||
− | + | <br> <br> | |
+ | Please note that protocols with | ||
+ | Q5 High-Fidelity DNA Polymerase may differ from protocols | ||
+ | with other polymerases. Conditions recommended below should be used for optimal | ||
+ | performance.<br> | ||
</div></div> | </div></div> | ||
Revision as of 11:49, 31 October 2017
Our research work
We are describing our research work. Below you can find the protocols we used.
Protocols
Main sponsors: