PCR Amplification#
General Protocol#
This protocol describes the general procedure to conduct PCR experiment. Optimal reaction conditions vary and need to be optimized.
Note
PCR reactions should be assembled in a DNA-free environment. Use of “clean” dedicated automatic pipettors and aerosol resistant barrier tips are recommended. Always keep the control DNA and other templates to be amplified isolated from the other components. [1]
Objective#
Amplify a DNA segment from tmplates.
Experiment Principle#
PCR is based on the natural process of DNA replication. It involves the enzymatic amplification of a specific DNA sequence using a pair of primers and a DNA polymerase enzyme. The process can produce millions to billions of copies of a particular DNA sequence from a small initial sample, making it possible to analyze DNA even when only a very small amount is available.
Materials and Reagents#
Reaction Buffer
DNA polymerase
dNTPs
Forward and Reverse Primers
Template DNA
Nuclease-free Water
Procedure#
Assemble reaction components
Attention
Assemble all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature.
Note
Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
Seting reaction thermal cycle
Step |
Temp Time |
|
|---|---|---|
Initial Denaturation |
95°C |
5 min |
Denaturation Cycle |
95°C |
30 s |
Anneal |
40-70°C (depended on Primer) |
30 s |
Extension Cycle |
72°C (depended on DNA polymerase) |
30 s (evaluate by extension speed) |
Final Extension |
72°C |
5 min |
Hold |
4-10°C |
Any |
Validate the PCR products
Use gel eletrophoresis to validate the PCR products.
References#
https://www.neb.com/en-sg/protocols/0001/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273