Library Preparation for 16S rRNA Sequencing Protocol

Polymerase Chain Reaction (PCR)

1. Prepare and clean the working station PCR hood or biosafety cabinet.
2. Thaw and keep the reagents on ice, vortex gently, and spin before use.
Prepare the PCR reaction in triplicates with the following reagents in PCR tubes or a 96-well plate. Nuclease-free water, 13.0 μL; PCR Master Mix, 10.0 μL; forward 515f primer (10 μM), 0.5 μL; reverse 806r primer (10 μM), 0.5 μL; and template DNA, 1.0 μL.
3. Turn on the thermocycler in advance for the instrument to warm up.
4. Prior to putting the tubes or plate into the thermocycler, spin them in a centrifuge for 290 × g for 1 min to bring the reagents together at the bottom of the tube or well.
5. Introduce the tubes or plate in the thermocycler and set up the following cycling conditions:
(a) 94 °C for 3 min.
(b) 94 °C for 45 s.
(c) 50 °C for 60 s.
(d) 72 °C for 90 s.
(e) Repeat steps (a–d) 35 times for total of 35 cycles.
(f) 72 °C for 10 min.
(g) Hold at 4 °C.
6. Once completed, samples can be safely stored at 4 °C or − 20 °C at this point. Avoid freeze-thaw cycles.
7. After amplification, pool triplicate samples into a single volume of 75 μL.

Agarose Gel Electrophoresis

1. Prepare 1.5% agarose into 1× TAE buffer in a flask. Volume will vary depending on the size of the gel box.
2. Microwave the flask to dissolve the agarose and let it boil for about 15 s. Make sure the solution is visually clear.
3. Put appropriate amount of SYBR Safe DNA Gel Stain (5 μL/50 mL) into the flask, and swirl gently to mix.
4. Slowly pour the mixture into the gel caster, use a pipet tip to remove any bubbles.
5. Place a comb of appropriate width into the gel, use a pipet tip to remove any bubbles, and let gel sit for about 25 min to solidify.
6. Once the gel has completely solidified, carefully remove the comb, lifting straight up. Do not damage the wells when removing the comb.
7. Place the gel tray into the gel box. (Check the orientation of the buffer chamber; the gel should run from black to red electrodes.)
8. Add 1× TAE buffer to barely immerse the gel but fully cover the wells.
9. Mix each sample with loading dye (e.g., 6× loading dye: 1 μL loading dye +5 μL PCR product).
10. Load DNA ladder and samples into the wells of the gel.
11. When finished loading samples including the no-template control (NTC) into the different wells of the gel, put the cover on the buffer chamber and plug the cords in to the electrophoresis box.
12. Turn on the electrophoresis box, and set the appropriate voltage and running time; set constant to volts.
13. When the gel has finished running, take the gel tray out of the buffer chamber, and carefully slide in a UV transilluminator or imager for visualization of the gel and the expected band of the amplicon.

Quantification and Normalization

1. Quantify the PCR amplicons by using a DNA quantification kit. We used Quant-iT PicoGreen dsDNA Assay Kit following manufacturer's instructions.
2. After the quantification of each sample pool equivalent amounts of 240 ng of amplicon from each sample into a sterile tube or tubes.

Cleanup PCR Product

1. Use a PCR cleanup kit for the pooled PCR products. QIAquick PCR purification kit is used following manufacturer's instructions.
2. Eluted cleaned pool library should be ready for submission to a sequencing facility. The best quality assurance and quality control (QA/QC) is to quantify the total library by quantitative PCR (qPCR).


1. Use KAPA Library Quantification Kit for Illumina platforms following manufacturer's instructions.
2. Once library is accurately quantified by qPCR, it should be ready for sequencing.
3. This library should be run in an Illumina MiSeq sequencer using a 2 × 250 paired-end chemistry.


  1. Maldonado J, Yaron J R, Zhang L, et al. Next-generation sequencing library preparation for 16S rRNA microbiome analysis after serpin treatment[J]. Serpins: Methods and Protocols, 2018: 213-221.
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