Oct 05, 2022
Sample preparation and lysis of homogenized malaise trap samples
- 1University of Duisburg-Essen, Aquatic Ecosystem Research
Protocol Citation: Dominik Buchner 2022. Sample preparation and lysis of homogenized malaise trap samples. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpjrmjgzp/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: September 29, 2022
Last Modified: October 05, 2022
Protocol Integer ID: 70661
Abstract
This protocol describes the steps of sample preparation and lysis before DNA extraction for the Malaise trap metabarcoding protocol of the LeeseLab.
Guidelines
Follow general lab etiquette. Wear gloves to prevent contaminating the samples. Clean the workspace before starting with 80% EtOH.
Materials
Materials required:
Below all materials needed for the protocol are listed. Vendors and part numbers are listed but interchangeable depending on thesupply situation.
Chemicals:
Tris ultrapure 99.9% Tris ultrapure 99.9%DiagonalCatalog #A1086.1000
Sodium chloride Sodium chloride Fisher ScientificCatalog #10112640
EDTA disodium salt EDTA disodium saltSigma AldrichCatalog #E5134-50G
SDS ultrapure Sodium dodecyl sulfateDiagonalCatalog #A1112.0500
Proteinase K Proteinase K7BioScienceCatalog #RP100B
Hydrochloric acid fuming 37% Hydrochloric acid fuming 37%Sigma AldrichCatalog #1003171011
Sodium hydroxide Sodium hydroxide - pelletsFisher ScientificCatalog #S/4920/60
Calcium chloride Calcium chloride 94%Carl RothCatalog #A119.1
Glycerol 87% Glycerol 87 % for molecular biologyPanreac AppliChemCatalog #A3739,1000
Labware:
2 mm dia zirconia beads Zirconia Beads 2 mm diaBioSpec ProductsCatalog #11079124zx
2 mL screwcap tubes T2 mL screwcap tubeSarstedtCatalog #72.693
Wide-bore tips ART Wide Bore Tip 1000 uLThermo Fisher ScientificCatalog #2079G
Disposable PES Bottle Top Filters Fisherbrand Disposable PES Bottle Top FiltersFisher ScientificCatalog #15973307
Stock solutions:
1 L Tris stock solution 1 Molarity (M) 7.5
- Add 121.14 g Tris ultrapure 99.9% to a beaker
- Adjust volume to 800 mL with ddH2O
- Adjust pH to 7.5 with HCl
- Adjust volume to 1 L
- Sterilize by filtering and store at Room temperature
1 L NaCl stock solution 5 Molarity (M)
- Add 292.2 g Sodium chloride to a beaker
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L EDTA stock solution 0.5 Molarity (M) 8
- Add 186.12 g EDTA disodium salt to a beaker
- Adjust volume to 1 L with ddH2O
- Adjust pH to 8 with sodium hydroxide
- Sterilize by filtering and store at Room temperature
1 L SDS stock solution 10 Mass Percent
- Add 100 g SDS ultrapure to a beaker
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L Proteinase K storage buffer (50 millimolar (mM) Tris , 3 millimolar (mM) CaCl2 50 % (v/v) glycerol )7.8
- Add 50 mL of1 Molarity (M) Tris stock solution 7.5
- Add 333 mg calcium chloride
- Add 500 mL of glycerol 87%
- Adjust volume to 900 mL with ddH2O
- Adjust pH to 7.8 with sodium hydroxide
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
Working solutions:
1 L TNES buffer (50 millimolar (mM) Tris , 400 millimolar (mM) NaCl , 20 millimolar (mM) EDTA ,0.5 Mass / % volume SDS ,7.5 )
- Add 50 mL of 1 Molarity (M) Tris stock solution 7.5
- Add 80 mL of 5 Molarity (M) NaCl stock solution
- Add 40 mL of 0.5 Molarity (M) EDTA stock solution 8
- Add 50 mL of 10 Mass / % volume SDS stock solution
- Adjust volume to 800 mL with ddH2O
- Adjust pH to 7.5 with HCl
- Sterilize by filtering and store at Room temperature
200 mL Proteinase K working solution (10 mg/mL Proteinase K )
- Dissolve 2 g of Proteinase K in 200 mL Proteinase K storage buffer
- Store at -20 °C
Safety warnings
Reagents are potentially damaging to the environment. Dispose waste responsibly.
Before start
Make sure all buffers are prepared before starting.
For each sample prepare a screwcap tube pre-filled with a few 2 mm zirconia beads.
Shake the sample well.
Transfer 800 µL of the small size fraction and 200 µL of the large size fraction to a 2 mL screwcap tube. It might be beneficial to use wide-bore tips or cut off the tip when using regular pipette tips.
11.000 x g, 00:03:00
3m
Remove as much ethanol as possible with a 1000 µL pipette.
Add 900 µL of TNES buffer and 100 µL of Proteinase K working solution. Vortex shortly.
Note
Depending on the amount of samples this can be prepared as a mastermix. We usually prepare TNES + Proteinase K in batches for 24 samples. Proteinase K tends to self-digest if the time for samples preparation takes too long.
Bead-beat for 00:02:00 at 2400 rpm
2m
Incubate 1400 rpm, 56°C, 00:20:00
Store at -20 °C until DNA extraction.