Team:Debrecen-Hungary/protocols/PCR

From 2010.igem.org

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'''Prepare Master mix 1'''
'''Prepare Master mix 1'''
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1. Add RNase free water to reach the final volume of 25 microliter
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1. Add RNase free water to reach the final volume of 25 microliter<br>
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<div style="float: right;"><html>
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<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object>
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</html></div><br><br>
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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>
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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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3. Add downstream primer to reach the final concentration of 300 nanomolar<Br><br>
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'''Prepare Master mix 2'''
 
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6. Add 19.25 microliter of RNase free water
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4. Add upstream primer in the same way.<br><br>
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7. Add 5 microliter of Expand High Fidelity Buffer with MgCl2
 
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8. Add 0.75 of Expand High Fidelity Enzyme Mix
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5. Add Template DNA which contains 10 to 250 nanogram complex DNA  <br><br>
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9. Vortex the components in both Master mixes
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'''Prepare Master mix 2'''<Br>
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10. Prepare the PCR tubes
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6. Add 19.25 microliter of RNase free water<Br>
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<div style="float: right;"><html>
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<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/j-hmoyMSc2Y" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/j-hmoyMSc2Y" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object>
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</html></div><br><Br>
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11. Divide the mix1 into the tubes in a desired volume
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7. Add 5 microliter of Expand High Fidelity Buffer with MgCl2<Br><Br>
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12. Then add the mix2 into the PCR tubes which contain mix 1
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8. Add 0.75 of Expand High Fidelity Enzyme Mix<Br><br>
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13. Set up the Thermocycler
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9. Vortex the components in both Master mixes<Br>
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<div style="float: right;"><html>
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<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/8_cafnNEqhU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8_cafnNEqhU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object>
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</html></div><br><br><br>
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10. Prepare the PCR tubes<Br><br>
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 +
11. Divide the mix1 into the tubes in a desired volume<Br>
 +
<div style="float: right;"><html>
 +
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/9fUZtcbOvqs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/9fUZtcbOvqs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object>
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</html><div><br><Br>
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12. Then add the mix2 into the PCR tubes which contain mix 1<Br><br>
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13. Set up the Thermocycler<br><br>
14. Place the PCR tubes into the thermocycler.  
14. Place the PCR tubes into the thermocycler.  
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<div style="float: right;"><html>
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<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/nDOxB_-aST0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/nDOxB_-aST0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object>
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</html><div><br><br>

Revision as of 20:11, 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
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