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Landscape of Interacting Microbes - PCR Protocols

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Colony PCR (27F/1492R) of the prokaryotic 16S rRNA gene

Primer	Sequence	Amplicon size
27F	AGAGTTTGATCATGGCTCA	1,465 bp
1492R	GGTTACCTTGTTACGACTT	1,465 bp

Master Mix

• Amplify the V4 region of the prokaryotic 16S rRNA gene from 1 µL of DNA or by touching a bacterial culture on a solid agar plate with a sterile pipette tip/toothpick and mixing into each PCR mixture

Reagent	Volume
PCR-grade water	9.1 µL
AmpliTaq Gold 360 Master Mix (2x)	12.5 µL
Primer 515F (10 µM)	1.2 µL
Primer 806R (10 µM)	1.2 µL
GC enhancer	1.0 µL
Total reaction volume	25.0 µL

Thermocycling conditions

Temperature	Time	Repeat
95 °C	10 min
95 °C	30 s	x29
55 °C	30 s	x29
72 °C	60 s	x29
72 °C	5 min
11 °C	hold

Post-PCR

• If there are <10 reactions, quantify each PCR product using the Qubit assay
• Alternatively, from each reaction, load 10 µL of PCR products on a 1% (wt/vol) 1xSB agarose gel and run gel at 170V for 45 minutes, then stain with ethidium bromide or SYBR Safe
• Purify PCR products with Zymo Research’s DNA Clean & Concentrator -10 or -25
• Quantify purified PCR products with Qubit
• Send PCR products to Eurofins Genomics for Sanger sequencing