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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

  Thread / Thread Starter Last Post Replies Views Forum
luisillo
05-14-2013 08:37 PM
by luisillo Go to last post
0 41 PCR - Polymerase Chain Reaction Forum
chinmayikaundinya
05-06-2013 12:48 PM
by muhammad ilyas Go to last post
6 1,706 PCR - Polymerase Chain Reaction Forum
claire5
04-19-2013 11:29 AM
by claire5 Go to last post
0 184 PCR - Polymerase Chain Reaction Forum
bmlmjone
04-15-2013 06:29 PM
by bmlmjone Go to last post
4 240 PCR - Polymerase Chain Reaction Forum
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