Jun 13, 2025
Historical Samples Extraction
- Dakota Betz1
- 1ucsd
- Rouse Lab
Protocol Citation: Dakota Betz 2025. Historical Samples Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb68x1lpk/v1
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: July 01, 2022
Last Modified: June 13, 2025
Protocol Integer ID: 65809
Keywords: historical sample, extraction, sample, rotary mixer
Disclaimer
Our protocols are constantly evolving and old versions will be deleted.
The documents here are not intended to be cited in publications
Abstract
Extraction protocol for historical samples. Requires overnight incubation and rotary mixer.
Attachments
sds_EDTA.pdf
56KB
Guidelines
Important Safety Note: Complete extraction steps in a fume hood and be very careful about any skin contact. Extraction buffer contains hazardous chemicals with inhalation hazards. Separate hazardous lab waste is needed for all waste products. See Safety Warnings below for more information.
Materials
Silica Magnetic Beads
guanidine isothiocyanate
1M Tris-HCl ph 7.5
0.2M EDTA
20% Sarkosyl
B-mercaptoethanol
PE Buffer
ddH2O
Troubleshooting
Safety warnings
Hazardous Chemicals Used (if applicable; SDS attached in Description):
- Guanidine isothiocyanate: causes severe skin burns and eye damage, harmful if swallowed, in contact with skin, or inhaled. If exposure occurs, immediately wash face, hands, and any exposed skin. Use in fumehood or well-ventilated area.
- 0.2M-0.5M EDTA: harmful if inhaled, best to use in fumehood or well-ventilated area.
- B-mercaptoethanol: acute toxicity and highly flammable. Avoid skin exposure or inhalation. Use in fumehood.
Safety Note
Complete extraction steps in a fume hood and be very careful about any skin contact. Extraction buffer contains hazardous chemicals. Separate hazardous lab waste is needed for all waste products. See Guidelines & Warnings for more information.
Extraction Buffer Preparation
In a 50 mL conical:
- 50 g guanidine isothiocyanate
- 5.3 mL 1M Tris-HCl pH 7.5
- 5.5 mL 0.2M EDTA
- 10.6 mL 20% Sarkosyl
- 1 mL B-mercaptoethanol
Add 50 mL of ddH2O.
Note
If using 0.5M EDTA, use 2.2 mL instead (remaining volume will be water) to dilute to 0.2M EDTA.
ALTERNATIVE Buffer Prep SCALED for Fewer Samples:
Volumes below are scaled to number of samples but you must make batches for at least 10--choose a total volume that is easy to check by eye for addition of ddH2O (amount for 1 sample: 0.2g guanidine isothiocyanate, 21.2uL 1M Tris-HCl, 22uL 0.2M EDTA, 42.4uL Sarkosyl, 4uL B-mercaptoethanol; bring volume up to 200uL).
- 0.2 g guanidine isothiocyanate (for 10 samples: 2g)
- 21.2 µL 1M Tris-HCl pH 7.5 (for 10: 212uL)
- 22 µL 0.2M EDTA or 8.8 µL 0.5M EDTA (for 10: 220uL)
- 42.4 µL 20% Sarkosyl (for 10: 424uL)
- 4 µL B-mercaptoethanol (for 10: 40uL)
Add 200 µL ddH2O (for 10: 2mL)
Day 1 - Incubation
Important Safety Note: Buffer mixture is flammable. Use caution if sanitizing with flame between specimens.
Note
Recommend prepping tissues in 1.5mL tubes first on separate lab bench, then transfer to fume hood and add extraction buffer. Work quickly to avoid further tissue degradation.
For each sample: place tissue in a 1.5 mL eppendorf tube with 200 uL of extraction buffer.
Incubate on hot plate at 55 °C Overnight .
Day 2 - Silica Bead Preparation
3m
Note
Note: Sillica Bead Preparation does not need to be performed in fume hood. Can prep at NGS lab bench instead.
Pipette or vortex G-Biosciences Silica Magnetic Beads thoroughly to re-suspend.
Transfer 20 µL of beads into a clean, labelled 1.5 mL eppendorf tube.
Note: This volume is scaled (amount for a single sample: 20uL).
Note
Up to 15 samples-worth of beads can be prepped in a single 1.5mL tube. However, more beads take longer to dry, etc. Consider prepping each tube individually for small numbers of samples.
If scaled to 8 samples: Use 160 uL of beads.
Place the tube on the magnetic stand for 30 - 60 seconds until clear: 00:00:30 - 00:01:00 .
Use a pipette to discard the supernatant.
1m 30s
Remove the tube from the magnetic stand. Add 100 µL ddH20 or Elution Buffer (do not use Elution Buffer for museum samples).
Resuspend the beads by pipetting or vortexing.
Note: This volume is scaled (amount for a single sample: 100uL)
Note
Elution buffer (if using): 10mM Tris-HCl, 1mM EDTA, pH 8.0
If scaled to 8 samples: Use 800 uL ddH20.
Place the tube on the magnetic stand for 30 - 60 seconds until clear: 00:00:30 - 00:01:00 .
Use a pipette to discard the supernatant.
1m 30s
Repeat steps 10-11 twice:
Remove tube from the magnetic stand. Add 20 µL ddH20.
Note: This volume is scaled (amount for a single sample: 20uL).
If scaled to 8 samples: Use 160 uL ddH20.
Pipette to resuspend. If applicable, aliquot bead mixture into number of tubes equal to number of samples you are extracting (number you scaled this protocol by). Each should have ~ 26.3uL if aliquotting from larger volume.
If scaled to 8 samples: Should have 8 tubes with ~20 uL of bead mixture in each.
Day 2 - Extraction
1h 57m
Reminder: Complete extraction steps that involve lysate in fume hood.
If any solid debris remains, centrifuge extraction 2 minutes: 12000 rpm, 00:02:00 .
2m
Add extraction lysate (liquid only, should be ~200uL) to each tube with 20uL of silica beads mixture. Discard any debris left over from centrifugation.
Add 200 uL of 100% Ethanol to lysate + beads. Gently mix and incubate in a rotary mixer for 15 minutes: 00:15:00 .
15m
Place tubes on magnetic stand for approximately 5 minutes, until clear: 00:05:00 .
Use a pipette to discard the supernatant.
5m
Pipette 200 uL of PE Buffer (Qiagen) and incubate in a rotary mixer for 10 minutes: 00:10:00 .
10m
Repeat steps 18 - 19 twice: . When ready to place on magnet stand for the final time (moving on to step 21), spin tubes down briefly (mini centrifuge) to get beads out of cap.
Keeping tubes on the magnetic stand, allow beads to air dry for 20 - 45 minutes: 00:30:00 - 00:45:00 .
1h 15m
Remove tubes from the magnet and elute beads in 20-50 uL of ddH2O. Use pipette to gently mix and use the elution H2O to encourage beads off the side of the tube. Incubate at 55 °C for 10 minutes: 00:10:00 at 55 °C .
10m
If needed, briefly spin tubes down (mini centrifuge) to get liquid out of caps.
Place tubes on magnetic stand. Wait until liquid is clear.
Use a pipette to move supernatant to a clean/new labeled tube, and discard the beads.