PCR PROTOCOL

Purpose – for amplifying gene of interest with a suitable primer. Allows the bands to be seen in
agarose gel. PCR at different temperature ranges and different concentration ranges allows
seeing at least 1 band formed in the agarose gel. For each forward and reverse primer, take the
average Tm for different temperature ranges.
Chemicals used – working concentration of primers, different concentration of working solutions
of primer, master mix, distilled water, dna template. 0.8% agarose gel, 1X TAE buffer, EtBr.
Materials – ice for thawing, autoclaves tips, stands, pcr tubes, PCR machine, electrophoresis
machine.
Procedure –
1. Thaw in ice all the reagents for the PCR.
2. Wipe the surface of table and pipettes with ethanol.
3. Use autoclaved tips and tubes for preparing the reactions as required.
4. Mix well in ice and spin.
5. Label and number.
6. Basic PCR settings –
a. 95 degrees – 2 mins
b. 95 degrees – 2 min 30 sec
c. 55 to 68 degrees – 30 sec
d. 72 degrees – 2 min 30 sec
e. 72 degrees – 30 sec
f. 4 degrees – store
7. Run in 0.8% agarose gel.
Results – you will get amplified dna bands in agarose gel. If all bands are seen till the dye front,
it means formation of primer dimer.
Reagents –
1. Pcr reagents
2. Agarose gel
3. buffers