Protocol #02
LABORATORY OF MOLECULAR PATHOLOGY
PCR for metagenomics
Using Platinum II polymerase for 16S, ITS and mcrA gene analysis Prep:
□ 01 Thaw an aliquot of PCR-grade water.
□ 02 Thaw an aliquot of Platinum II polymerase, keep it on ice.
□ 03 Thaw 10 U.M primer aliquots, keep them on ice.
□ 04 Dilute reasonable volumes of primers you will need to 2.5 u.M
i.e. 2 ju/. of primer per sample plus excess
□ 05 Calculate the master mix composition you will need for all the samples together.
□ 06 Compose the master mix for the whole PCR reaction. Don't forget to include negative controls, where instead of DNA you add pure water.
Master mix	lx	x
Polymerase 2x	10,0 uL	
FW primer 2.5 uM	2,0 uL	-
REV primer 2.5 uM	2,0 uL	-
DEPC-H20	9,0 uL	
DNA sample	2,0 uL	-
□ 07 Pipette 19 u.L of the master mix into the wells.
□ 08 Pipette 2 u.L of the 2.5 U.M forward primer. One row, one primer.
□ 09 Pipette 2 u.L of the 2.5 u.M reverse primer. One column, one primer.
□ 10 Pipette 2 u.L of your DNA sample into the well
□ 10 Cover the wells with strong adhesive foil. Make sure it adhered properly.
□ 11 Do a quick vortex to mix the reactions and quick spin.
□ 12 Put the plate into the cycler and run the following programme. You need to change the ramp rate of the annealing to 60 %.
Amplification programme	Temperature	Time
Initial denaturation	95 °C	3 min
		
Denaturation	95 °C	45 s
Annealing	52 °C	60 s 35x
Extension	72 °C	90s
		
Final extension	72 °C	5 min
12 °C Hold