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Isolate_16S.md

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Pure culture isolate DNA extraction and 16S rRNA sequencing protocol

Version 1.3: 4 Jab, 2022

1. Scope and Principle

This protocol is intended for the identification of pure culture isolates from an agar plate or broth culture. Genomic DNA is extracted in a quick and dirty way using Instagene (Biorad) although any genomic isolation prep could be used instead. PCR is then performed, purified and sent for paired Sanger sequencing.

2. Materials

  • Instagene Matrix (Biorad #732-6030)
  • Wide bore 200 µL pipette tips (or clean scissors to cut tip off end of normal tip)
  • 1.5 mL nuclease-free microcentrifuge tubes
  • nuclease-free water or PBS
  • 200 µL PCR tubes
  • Waterbath/heatblock/PCR thermocycler capable of 56˚C and 100˚C
  • Amplitaq Gold 360 Master Mix (Life Tech/Thermofisher 4398881)
  • 2µM 8F (pA) primer (5-AGAGTTTGATCCTGGCTCAG-3)
  • 2µM 1542R (pH) primer (5- AAGGAGGTGATCCAGCCGCA-3)
  • Purelink PCR purification kit (Life Technologies #K3100-01; or Qiagen equivalent)
  • (optional) 2µM 515F primer (5-GTGYCAGCMGCCGCGGTAA-3)
  • (optional) 2µM 806RB primer (5-GGACTACNVGGGTWTCTAAT-3)
  • (optional) AMPure XP bead (Beckman coulter A63881)
  • (optional) Freshly prepared 70% EtOH
  • (optional) Magnetic capture plate

3. DNA Extraction

  • Pick a single colony from a freshly prepared culture plate and resuspend in 1mL of PBS or sterile H2O in a microcentrifuge tube OR Transfer 200ul from a broth culture to a microcentrigue tube.
  • Centrifuge at ~10,000 xg for 1 minute.
  • Remove the supernatant and resuspend in 100 µL of Instagene matrix (matrix should be well mixed and use wide bore tips)
  • Mix thoroughly by vortexing
  • Incubate at 56˚C for 30 minutes
  • Mix thoroughly by vortexing
  • Incubate at 100˚C for 8 minutes
  • Mix thoroughly by vortexing
  • Centrifuge for 3 minutes at max (~16,000 xg)
  • Transfer 60 µL of the supernatant containing gDNA to a new microcentrifuge tube. This is optional and supernatant can be used directly for PCR.
  • Store gDNA at -20˚C for long term storage or 4˚C for short term.

4. PCR

  • Prepare a master mix for PCR as outlined in Table 4.1.
  • Add 2 µL of gDNA for a total reaction volume of 20 µL. Be sure to include a reaction with 2 µL of H2O as no template control (NTC).
  • Pop spin to remove any air bubbles.
  • Run the PCR using the PCR cycle parameters indicated in Table 4.2
Table 4.1 PCR Master Mix
Component Stock Concentration Final Concentration per 20µL rxn
Amplitaq Gold 2x 1x 10 µL
8F 2µM 400 nM 4 µL
1542R 2µM 400nM 4 µL
gDNA variable variable 2µL
Total 20 µL
Table 4.2 PCR Cycles
Cycle Temperature (˚C) Time
Initial Denaturation 95 10 min
30 cycles:
Denature 95˚C 30 sec
Anneal 55˚C 30 sec
Extend 72˚C 60 sec
Holding 4˚C Hold 0 sec

5. Reaction cleanup

Option A: AMPure XP beads

This protocol works better for a large number of samples being processed.

  • Add 18 µL AMPure XP beads (make sure well suspended) to 20 µL PCR reaction (0.9x volume beads)
  • Mix by pipetting and capture for ~5 min on magnetic rack
  • Keeping tubes on magnetic rack, remove supernatant and replace with 200 µL fresh 70% ethanol.
  • Keeping tubes on magnetic rack, remove supernatant and replace with 200 µL fresh 70% ethanol.
  • Keeping tubes on magnetic rack, remove supernatant.
  • Pop spin to bring down any ethanol on the sides. Capture on stand.
  • Use 10µL pipette to remove any trace EtOH on bottom of rack.
  • Air dry ~10 min.
  • Add 25 µL water and pipette to mix. This step elutes the DNA off the beads. Capture on magnetic rack ~2-5 minutes.
  • Remove 20 µL of eluted DNA to fresh tube and discard bead tubes.

Option B: Purelink PCR cleanup

If only a few samples are being processed this will be faster. The Qiagen PCR cleanup kit can be used interchangeably with the same protocol and volumes.

  • Add 200 µL of buffer B2 to 50 µL of the reaction (4 volumes).
  • Transfer 250 µL of the mixture to a spin column and centrifuge at maximum speed for 1 minute.
  • Discard flow through and add 650 µL of wash buffer W1. Centrifuge at maximum speed for 1 minute.
  • Discard flow through and recentrifuge for 3 minutes.
  • Transfer spin column to fresh nuclease free microcentrifuge tube and add 50 µL of Elution buffer (Tris-HCl, TE, H2O will all work as well).
  • Incubate at room temperature for 1 minute and then centrifuge for 2 minutes at maximum speed.
  • Dispose of spin column and keep eluted DNA.

6. QC

  • Use a nanodrop or qubit to quantify products as well as NTC. Yield per this protocol should yield ~20-40 ng/µL product with <2 ng/µL in the NTC.
  • Dilute PCR products to 10 ng/µL
  • (optional) Prepare and run a 1% agarose gel using TAE or TBE buffer using 10 µL of PCR reaction. Confirm presence of ~1,500 bp band and the absence of product in the NTC.

7. Sequencing

*GeneWiz requests sequencing reactions in strip tubes if <48 samples. If using strip tubes, label the first and last tube with the corresponding ID from the genewiz order confirmation. If a full length sequence is desired, both 8F and 1543R can be used for sequencing in two different reactions, otherwise just use 8F.

  • Using GeneWiz account found on slack channel, create a sequencing request for premixed sequencing. Select Sanger Sequencing with the following options: DNA Type = PCR Product - Purefied; Service Type = Premixed; # of Samples = 2; Purification Type = Other Ampure XP. The order confirmation will include a specified layout of strip tubes or plates for each combination of sample and primer.
  • For each sequencing reaction, place 5 µL of 10 ng/µL product into a strip tube and add 10 µL of 2 µM 8F (or 1543R but not both!)). Do not add forward and reverse primers to the same tube.
  • Drop off samples in drop box.

8. Cleaning and interpreting sequence

  • When sequence results are available (usually next day), download .ab1 files.
  • Using 4 Peaks (mac) or FinchTV (windows) trim trace to remove any Ns, resolving them by manually inspecting the electropherogram where possible.
  • If you sequenced from both sides: overlap sequences using a program such as CAP3.
  • Nucleotide BLAST against NCBI nr and NCBI 16S reference sequences excluding matches to uncultured material.
  • A species level identification should involve a >97% match over the entire length of the product to a defined species. If there are multiple equal matches (for example to Lactobacillus casei, and Lactobacillus rhamnosus), it will not be possible to resolve the species from 16S rRNA alone. Try excluding the species your top hit is against and see the nearest match.