IGEM:University of Debrecen: Polymerase Chain Reaction (PCR)

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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