Team:Debrecen-Hungary/protocols/PCR

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1. Add RNase free water to reach the final volume of 25 microliter
1. Add RNase free water to reach the final volume of 25 microliter
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2. Add 1 microliter of PCR grade nucleotide mix
 
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3. Add downstream primer to reach the final concentration of 300 nanomolar
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2. Add 1 microliter of PCR grade nucleotide mix <br><br><br>
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3. Add downstream primer to reach the final concentration of 300 nanomolar<Br><br>
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4. Add upstream primer in the same way.
4. Add upstream primer in the same way.
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5. Add Template DNA which contains 10 to 250 nanogram complex DNA 
 
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'''Prepare Master mix 2'''
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5. Add Template DNA which contains 10 to 250 nanogram complex DNA  <br><br><br>
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6. Add 19.25 microliter of RNase free water
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'''Prepare Master mix 2'''<Br><br><br>
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7. Add 5 microliter of Expand High Fidelity Buffer with MgCl2
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6. Add 19.25 microliter of RNase free water<Br>
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8. Add 0.75 of Expand High Fidelity Enzyme Mix
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7. Add 5 microliter of Expand High Fidelity Buffer with MgCl2<Br><br><br>
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9. Vortex the components in both Master mixes
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8. Add 0.75 of Expand High Fidelity Enzyme Mix<Br><br><Br>
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10. Prepare the PCR tubes
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9. Vortex the components in both Master mixes<Br>
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11. Divide the mix1 into the tubes in a desired volume
 
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12. Then add the mix2 into the PCR tubes which contain mix 1
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10. Prepare the PCR tubes<Br><br><br>
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13. Set up the Thermocycler
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11. Divide the mix1 into the tubes in a desired volume<Br><br><br>
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14. Place the PCR tubes into the thermocycler.  
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12. Then add the mix2 into the PCR tubes which contain mix 1<Br>
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13. Set up the Thermocycler<br><br><br>
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14. Place the PCR tubes into the thermocycler.<br>
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<br><br><br><br>
==An example program==
==An example program==
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• Denature: 96°C, 5 mins
• Denature: 96°C, 5 mins
This denatures any double stranded DNA.
This denatures any double stranded DNA.
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Thermocycling
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Thermocycling<br>
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• No. of cycles: 25
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• No. of cycles: 25<br>
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• Denature: 96°C, 1 min
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• Denature: 96°C, 1 min<br>
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• Anneal: 55°C, 30 secs
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• Anneal: 55°C, 30 secs<br>
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• Elongate: 72°C, 1 min
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• Elongate: 72°C, 1 min<Br>
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Termination
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Termination<br>
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• Elongate: 72°C, 5 mins
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• Elongate: 72°C, 5 mins<br>
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• Hold: 4°C, until removed from machine
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• Hold: 4°C, until removed from machine<br>
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3. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, and Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986; 51 Pt 1 263-73.pmid:3472723. PubMed HubMed [Mullis-1986]
3. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, and Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986; 51 Pt 1 263-73.pmid:3472723. PubMed HubMed [Mullis-1986]
first public presentation on PCR
first public presentation on PCR
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==Links==
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[http://www.youtube.com/user/debrecenigem2010#p/u/11/vIbKpzlOAXA Video I & II]
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[http://www.youtube.com/user/debrecenigem2010#p/u/10/j-hmoyMSc2Y Video III]
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[http://www.youtube.com/user/debrecenigem2010#p/u/9/8_cafnNEqhU Video IV]
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[http://www.youtube.com/user/debrecenigem2010#p/u/8/9fUZtcbOvqs Video V]
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[http://www.youtube.com/user/debrecenigem2010#p/u/7/nDOxB_-aST0 Video VI]

Latest revision as of 21:07, 26 October 2010



Contents

Overview

PCR is an acronym for polymerase chain reaction. It is a method for amplifying DNA in vitro.

Reagents

• Expand High Fidelity Buffer with MgCl2

• PCR grade nucleotide mix MgCl 2

• Upstream primer

• Downstream primer

• Template DNA

• RNase free water

• Expand High Fidelity Enzyme Mix

Equipment

• Thermocycler

• Reaction tubes

Step

Prepare Master mix 1

1. Add RNase free water to reach the final volume of 25 microliter



2. Add 1 microliter of PCR grade nucleotide mix


3. Add downstream primer to reach the final concentration of 300 nanomolar


4. Add upstream primer in the same way.



5. Add Template DNA which contains 10 to 250 nanogram complex DNA


Prepare Master mix 2


6. Add 19.25 microliter of RNase free water



7. Add 5 microliter of Expand High Fidelity Buffer with MgCl2


8. Add 0.75 of Expand High Fidelity Enzyme Mix


9. Vortex the components in both Master mixes



10. Prepare the PCR tubes


11. Divide the mix1 into the tubes in a desired volume


12. Then add the mix2 into the PCR tubes which contain mix 1



13. Set up the Thermocycler


14. Place the PCR tubes into the thermocycler.





An example program

Program a standard thermocycler to run the reaction using the following parameters: Initial denaturation • Denature: 96°C, 5 mins This denatures any double stranded DNA. Thermocycling
• No. of cycles: 25
• Denature: 96°C, 1 min
• Anneal: 55°C, 30 secs
• Elongate: 72°C, 1 min
Termination
• Elongate: 72°C, 5 mins
• Hold: 4°C, until removed from machine


References

1. Arezi B, Xing W, Sorge JA, and Hogrefe HH. Amplification efficiency of thermostable DNA polymerases. Anal Biochem 2003 Oct 15; 321(2) 226-35. pmid:14511688. PubMed HubMed [Arezi-AnalBiochem-2003]

2. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, and Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science 1985 Dec 20; 230(4732) 1350-4. pmid:2999980. PubMed HubMed [Saiki-Science-1985] original paper on PCR

3. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, and Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986; 51 Pt 1 263-73.pmid:3472723. PubMed HubMed [Mullis-1986] first public presentation on PCR


Links

[http://www.youtube.com/user/debrecenigem2010#p/u/11/vIbKpzlOAXA Video I & II]

[http://www.youtube.com/user/debrecenigem2010#p/u/10/j-hmoyMSc2Y Video III]

[http://www.youtube.com/user/debrecenigem2010#p/u/9/8_cafnNEqhU Video IV]

[http://www.youtube.com/user/debrecenigem2010#p/u/8/9fUZtcbOvqs Video V]

[http://www.youtube.com/user/debrecenigem2010#p/u/7/nDOxB_-aST0 Video VI]

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