Team:HKUST/Protocols/PCR

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(New page: 2. PCR Purpose: To amplify a specific piece of DNA out from the whole. Materials: ddH2O, 10X ThermoPol Buffer, 1mM dNTP, Forward primer, Reverse Primer, Taq or Vent DNA polymerase, ...)
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2. PCR
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  Purpose: To amplify a specific piece of DNA out from the whole.  
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<head>
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  Materials: ddH2O, 10X ThermoPol Buffer, 1mM dNTP, Forward primer, Reverse Primer, Taq or Vent DNA polymerase, DNA template
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<title>Salt and Soap template</title>
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<div class="contentlist"> <h3>a</h3>
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<p>PCR</p>
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<p>  Purpose: To amplify a specific piece of DNA out from the whole. </p>
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Materials: ddH2O, 10X ThermoPol Buffer, 1mM dNTP, Forward primer, Reverse Primer, Taq or Vent DNA polymerase, DNA template
               Choice of DNA polymerase: Taq gives higher rate of mutation compared with Vent.
               Choice of DNA polymerase: Taq gives higher rate of mutation compared with Vent.
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                                         Taq gives sticky ends after PCR, while Vent gives blunt ends.
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                                         Taq gives sticky ends after PCR, while Vent gives blunt ends. <br><br>
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  Procedure:  
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  a.Add 10 μL water to make it a 20 μL reaction.
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<p>Procedure: </p>
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  b.Add 2 μL buffer, 4 μL dNTP, 1 μL forward primer (5 μM), 1 μL reverse primer (5 μM), 1 μL DNA template, 1 μL DNA polymerase
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1. Add 10 μL water to make it a 20 μL reaction. <br>
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  c.Vortex and then spin down.
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2. Add 2 μL buffer, 4 μL dNTP, 1 μL forward primer (5 μM), 1 μL reverse primer (5 μM), 1 μL DNA template, 1 μL DNA polymerase <br>
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  d.Put it into the PCR machine and set the program.
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3. Vortex and then spin down.<br>
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     Program
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4. Put it into the PCR machine and set the program.<br>
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     (1) Initial denaturation  95 °C  4 mins
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     Program<br>
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     (2) Run 25-30 cycles of:
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     (1) Initial denaturation  95 °C  4 mins<br>
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         Denaturation          95 °C  30 secs
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     (2) Run 25-30 cycles of:<br>
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         Annealing                    30 secs  
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         Denaturation          95 °C  30 secs<br>
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                                 Temperature is depended on melting temperature of primer.  
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         Annealing                    30 secs <br>
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                                 Lower Tm-5°C, for Taq; Lower Tm +3°C for Vent
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                                 Temperature is depended on melting temperature of primer. <br>
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         Extension              72 °C  30 secs per 500bp PCR product length
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                                 Lower Tm-5°C, for Taq; Lower Tm +3°C for Vent<br>
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     (3) Final extension        72 °C  3~5 mins
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         Extension              72 °C  30 secs per 500bp PCR product length<br>
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  Tips:  
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     (3) Final extension        72 °C  3~5 mins<br>
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  •The DNA polymerase is easy to be denatured, so do not leave it at room temperature. It is highly recommended to use an ice holder or do the adding in the fridge.
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<p> Tips: </p>
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The DNA polymerase is easy to be denatured, so do not leave it at room temperature. It is highly recommended to use an ice holder or do the adding in the fridge.
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<ul>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel
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electrophoresis</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to
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yeast</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA
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extraction</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li>
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Revision as of 15:28, 19 October 2009

Salt and Soap template

a

PCR

Purpose: To amplify a specific piece of DNA out from the whole.

Materials: ddH2O, 10X ThermoPol Buffer, 1mM dNTP, Forward primer, Reverse Primer, Taq or Vent DNA polymerase, DNA template Choice of DNA polymerase: Taq gives higher rate of mutation compared with Vent. Taq gives sticky ends after PCR, while Vent gives blunt ends.

Procedure:

1. Add 10 μL water to make it a 20 μL reaction.
2. Add 2 μL buffer, 4 μL dNTP, 1 μL forward primer (5 μM), 1 μL reverse primer (5 μM), 1 μL DNA template, 1 μL DNA polymerase
3. Vortex and then spin down.
4. Put it into the PCR machine and set the program.
Program
(1) Initial denaturation 95 °C 4 mins
(2) Run 25-30 cycles of:
Denaturation 95 °C 30 secs
Annealing 30 secs
Temperature is depended on melting temperature of primer.
Lower Tm-5°C, for Taq; Lower Tm +3°C for Vent
Extension 72 °C 30 secs per 500bp PCR product length
(3) Final extension 72 °C 3~5 mins

Tips:

The DNA polymerase is easy to be denatured, so do not leave it at room temperature. It is highly recommended to use an ice holder or do the adding in the fridge.
HKUST

Retrieved from "http://2009.igem.org/Team:HKUST/Protocols/PCR"