PCR Amplification of 16S rRNA Genes
Wong
To test genomic DNA for amplification by PCR, set up 10 µL pilot reactions. Recipe can be
scaled up for preparative PCR.
To ensure adequate volume of reagents, prepare a PCR MasterMix for 1 more reaction than you
need. The following example is a pilot PCR experiment for one or ten genomic DNA samples.
11 x 10 µL reactions = 110 µL total volume
Genomic template DNA = 0.5 µL per reaction
Master Mix = 9.5 µL per reaction
Total Master Mix volume = 11 x 9.5 µL = 104.5
Thaw solutions quickly (hand warmth), and vortex briefly. Prepare master mix under PCR-clean
conditions (sterile hood with gloves and sleeve protectors), with all reagents kept cold (on ice
or cold box)
MASTER MIX (stock reagents from Qiagen)
1 rxn
10x PCR Buffer
1
BSA (1 mg/mL non-acetylated stock)
0.8
8.8
dNTP mix (10 mM each dNTP)
0.2
2.2
63F/1387R primer mix (5 pmol/µL each in mix)
0.8
8.8
Q solution (optional; test +/- Q effect)
(2.2)
Taq DNA polymerase
0.04
H2O
bring to 9.5 total
________
Total Master Mix volume
9.5 µL
11 rxns
11
(22)
0.5
bring to 104.5 total
_______
104.5 µL
Add Master Mix and genomic DNA to 200 µL PCR tubes kept on cold rack.
Each Sample
Master Mix
9.5 µL
Genomic template DNA
0.5 µL
PCR CONDITIONS
Start up thermocycler ahead of time to allow lid to prewarm.
Bio-Rad iCycler or MyCycler thermocycler
95°C, 4 min preheat; 30 cycles of 94°C, 30 sec / 55°C, 30 sec / 72°C, 2 min; 72°C, 7 min final
extension; 4°C hold.
Run usually takes about 3 hours to complete
ASSESSING PCR PRODUCTS
For gel analysis of PCR products, prepare a 0.7% agarose gel, 0.5x Lithium Borate (LB) running
buffer. 16S rDNA product is about 1.3 kb in size. Run BioLine Ladder I as size markers.