PCR protocol

From Molecular Biology Wiki

Standard PCR Protocol

Also see PCR - polymerase chain reaction.

Standard Basic PCR protocol

For a PCR with 25 μl reaction volume add the following to your pcr master mix.

Prepare the following reaction mix on ice:

For a 25μl reaction volume: Component Volume Final Conc.
PCR Master Mix, 2X 12.5μl 1X
Forward Primer, 10μM 0.25–2.5μl 0.1–1.0μM
Reverse Primer, 10μM 0.25–2.5μl 0.1–1.0μM
DNA template 1–5μl <250ng
Nuclease-Free Water to 25μl

Thermocyler Settings:

Step 1) Denaturation

A 2-minute initial denaturation step at 95°C. (Subsequent denaturation steps can be between 30 seconds and 1 minute).

Step 2) Annealing Approximately 5°C below the calculated melting temperature of the primers (Tm). Usually 30 seconds to 1 minute.

Step 3) Extension The extension reaction is performed at the optimal temperature for Taq DNA Polymerase which is from 68–74°C depending on the Taq you are using (see manufacturers enzyme information).

Cycle number: This is anywhere from 25–35 PCR cycles.

Make sure to hold the tubes at 4°C after cycling to keep the reaction cold.

PCR Protocols Discussion

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