Team:Debrecen-Hungary/protocols/PCR
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(New page: ==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 nucleotid...) |
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==Overview== | ==Overview== | ||
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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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+ | <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> | ||
+ | </html></div><br> | ||
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- | 3. Add downstream primer to reach the final concentration of 300 nanomolar | + | 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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+ | <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> | ||
+ | </html></div><br> | ||
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- | + | 5. Add Template DNA which contains 10 to 250 nanogram complex DNA <br><br><br> | |
- | + | '''Prepare Master mix 2'''<Br><br><br> | |
- | + | 6. Add 19.25 microliter of RNase free water<Br> | |
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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> | ||
+ | </html></div><br><br> | ||
- | + | 7. Add 5 microliter of Expand High Fidelity Buffer with MgCl2<Br><br><br> | |
- | + | 8. Add 0.75 of Expand High Fidelity Enzyme Mix<Br><br><Br> | |
- | + | 9. Vortex the components in both Master mixes<Br> | |
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+ | <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> | ||
+ | </html></div><br> | ||
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- | + | 10. Prepare the PCR tubes<Br><br><br> | |
- | + | 11. Divide the mix1 into the tubes in a desired volume<Br><br><br> | |
- | + | 12. Then add the mix2 into the PCR tubes which contain mix 1<Br> | |
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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> | ||
+ | </html></div><br><br> | ||
+ | 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. | ||
- | Thermocycling | + | Thermocycling<br> |
- | • No. of cycles: 25 | + | • No. of cycles: 25<br> |
- | • Denature: 96°C, 1 min | + | • Denature: 96°C, 1 min<br> |
- | • Anneal: 55°C, 30 secs | + | • Anneal: 55°C, 30 secs<br> |
- | • Elongate: 72°C, 1 min | + | • Elongate: 72°C, 1 min<Br> |
- | Termination | + | Termination<br> |
- | • Elongate: 72°C, 5 mins | + | • Elongate: 72°C, 5 mins<br> |
- | • Hold: 4°C, until removed from machine | + | • 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
OverviewPCR 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 StepPrepare Master mix 1 1. Add RNase free water to reach the final volume of 25 microliter
3. Add downstream primer to reach the final concentration of 300 nanomolar
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
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 programProgram 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
References1. 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] |