Oct 06, 2025

Public workspaceBONCAT_DECODE

This protocol is a draft, published without a DOI.
  • Tim Goodall1,
  • Susheel Bhanu Busi2
  • 1UKCEH;
  • 2UK Centre for Ecology and Hydrology
Susheel Bhanu Busi
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Protocol Citation: Tim Goodall, Susheel Bhanu Busi 2025. BONCAT_DECODE. protocols.io https://protocols.io/view/boncat-decode-c88nzzve
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 14, 2024
Last Modified: October 06, 2025
Protocol Integer ID: 95214
Keywords: decode analysis
Abstract
DECODE analysis
Protocol materials
ReagentTween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P-7949
Troubleshooting
Sample prep and HPG incubation
17m
  1. Transfer 1 g of soil to a 50 ml falcon tube
  2. Add 5 ml 1X PBS and 5 sterile glass beads
  3. Vortex for Duration00:02:00
  4. Remove roots and add 20 ml 1X PBS
  5. Add Amount32 µL of 100 mg/ml HPG
  6. Incubate for Duration00:15:00 at room temperature
17m
Cell separation and fixation
1h
  1. Add 5 ml of 0.2% ReagentTween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P-7949 in 1X PBS {see Probing_active_fraction_of_soil_microbiomes}
  2. Vortex forDuration00:05:00
  3. Centrifuge at 500 g for 55 minutes {yes, you read it right - Duration00:55:00 }
  4. Collect supernatant into a fresh falcon tube
  5. Add 1:1 100% EtOH (final volume: Concentration50 % (v/v) )
  6. Store at Temperature-20 °C for downstream processing

1h
Click reaction and staining
  1. Perform the `click reaction` as described in protocol by Moss et al.
  2. The collected cell population includes both active and inactive fractions (if sample in 50% ETOH dilute 2:3 in PBS)
  3. Staining with SYBR Green == "total population" (if sample in 50% ETOH dilute 1:49 in PBS prior to staining)
  4. HPG count == "active population"

Note
Run HPG count prior to SYBR green due to potential crossover events


Below are steps for field based HPG incorporation and preservation
Alternative approach for field base HPG incubation and cell fixation
1. Transfer up to 1g of soil/rhizosphere material to 50ml centrifuge tube
2. Add PBS at 25ml per gram material and 5 glass beads (e.g. if 0.4g add Amount10 mL )
3. Vortex Duration00:02:00
4. For rhizosphere samples remove roots by tipping slurry into new tube
5. Add Amount32 µL of 100 mg/ml HPG per gram material (e.g. if 0.4g add Amount12.8 µL )
6. Incubate for Duration00:15:00 at room temperature
7. Fix cells by 1:1 addition of 4% PFA (final concentration Concentration2 % (v/v) )
8. Briefly mix and store refrigerated


17m
Cell separation, post field collection
1h
1. Add 0.2% Tween-20 in PBS at 5ml per gram material (e.g. if 0.4g add 2ml)
2. Vortex forDuration00:05:00
3. Centrifuge at 500 g for 55 minutes {yes, you read it right - Duration00:55:00 }
4. Collect supernatant into a fresh falcon tube
1h
Click reaction
Refer to Moss et al