Feb 02, 2022
Version 1
HMW DNA extraction for amphipods V.1
- 1Genoscope/CEA
- Benoit
Protocol Citation: Benoît Vacherie, Karine Labadie 2022. HMW DNA extraction for amphipods. protocols.io https://dx.doi.org/10.17504/protocols.io.b4iwqufe
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 01, 2022
Last Modified: February 02, 2022
Protocol Integer ID: 57654
Keywords: HMW, Nanopore, Amphipods, DNA Extraction,
Abstract
Protocol adapted from Qiagen's genomic DNA handbook protocol for the HMW extraction of live or flash-freezed amphipods.
Guidelines
To preserve large DNA sizes, never use Vortex and only use Wide-Bore tips.
Materials
Reagent :
Genomic-tip 20/GQiagenCatalog #ID: 10223
Buffer G2QiagenCatalog #1014636
Buffer QBT
Buffer QF
Buffer QC
RNase AQiagenCatalog #19101
Proteinase K (2 ml)QiagenCatalog #19131
2-propanol Sigma Aldrich
Ethanol 70% [Note: freshly prepared]
Tris-EDTA (TE) buffer pH 8.0 1X
Consumables :
2 mL Eppendorf
1.5 mL Eppendorf tubes
15 mL Falcon tubes
ART Wide-Bore tips 1000 µlCatalog #2079GPK
ART Wide-Bore tips 200µlCatalog #2069GPK
Equipment:
Equipment
Dounce Homogenizer, 2mL
NAME
Tissue Grinder
TYPE
Kimble
BRAND
885300-0002
SKU
LINK
2 mL with Pestles A and B
SPECIFICATIONS
Equipment
ThermoMixer
NAME
Benchtop Incubator
TYPE
Eppendorf
BRAND
5382000023
SKU
LINK
Any heat block will suffice
SPECIFICATIONS
Equipment
Centrifuge 4K15
NAME
Sigma
TYPE
Centrifuge
BRAND
BLE1700081
SKU
Protocol materials
RNase AQiagenCatalog #19101
Buffer QBT
2 mL Eppendorf
ART Wide-Bore tips 200µlCatalog #2069GPK
Buffer QF
Buffer QC
2-propanol Merck MilliporeSigma (Sigma-Aldrich)
Ethanol 70% [Note: freshly prepared]
1.5 mL Eppendorf tubes
15 mL Falcon tubes
ART Wide-Bore tips 1000 µlCatalog #2079GPK
Tris-EDTA (TE) buffer pH 8.0 1X
Genomic-tip 20/GQiagenCatalog #ID: 10223
Buffer G2QiagenCatalog #1014636
Proteinase K (2 ml)QiagenCatalog #19131
RNase AQiagenCatalog #19101
Buffer G2QiagenCatalog #1014636
2 mL Eppendorf
Proteinase K (2 ml)QiagenCatalog #19131
2 mL Eppendorf
15 mL Falcon tubes
Genomic-tip 20/GQiagenCatalog #ID: 10223
Buffer QBT
Buffer QC
Buffer QF
2-propanol Merck MilliporeSigma (Sigma-Aldrich)
Ethanol 70% [Note: freshly prepared]
1.5 mL Eppendorf tubes
Tris-EDTA (TE) buffer pH 8.0 1X
Tissue homogenization
Tissue homogenization
10m
10m
Prepare the lysis buffer by adding 3 µL of RNase A (100 mg/mL) to 1.5 ml of Buffer G2 per sample.
RNase AQiagenCatalog #19101
Buffer G2QiagenCatalog #1014636
place live or flash-freezed amphipod in 2ml douncer and add 1 ml of lysis buffer
gently up and down 10 times with the piston
transfer the lysate to a 2 ml tube
2 mL EppendorfContributed by users
rinse the douncer with 0.5 ml lysis buffer and transfer to the 2 ml tube
Lysis
Lysis
2h 30m
2h 30m
Incubate for 30 minutes at 37°C.
37 °C 00:30:00
30m
Add 75 µL of Proteinase K (20 mg/mL) and incubate with inversion at 50°C for 2 hours.
Proteinase K (2 ml)QiagenCatalog #19131
02:00:00 50 °C
2h
Gently transfer in a new 2 ml tube the supernatant with a 1000 µl wide-bore tip, avoiding touching the pelleted debris by gravity.
2 mL Eppendorf
DNA binding and washing
DNA binding and washing
1h
1h
Place a genomic-tip 20G column on a 15 ml tube
15 mL Falcon tubesContributed by users
Genomic-tip 20/GQiagenCatalog #ID: 10223
Equilibrate the column with 1 mL Buffer QBT. Wait for the Genomic-tip to drain by gravity.
Buffer QBTContributed by users
Carefully apply the lysate to the Genomic-tip with a 1000 µl wide-bore tip. Wait for the Genomic-tip to drain by gravity.
Wash the QIAGEN Genomic-tip with 3 x 1 mL Buffer QC.
Buffer QCContributed by users
Elute the DNA into a new 15 mL tube with 2 x 1 mL of Buffer QF prewarm to 50°C
Buffer QFContributed by users
DNA recovery
DNA recovery
1h 50m
1h 50m
Add 1.4 mL of room-temperature isopropanol to the éluate, mix by inversion about 10 times, and centrifuge for 30 min at 5,500g at 4°C to pellet DNA. set centrifuge deceleration speed to level3
2-propanol Sigma Aldrich
5500 x g, 4°C, 00:30:00 , Acc 9 / Dec 3
30m
Gently remove the supernatant using a P1000
Gently resuspend the pellet with 1 ml of cold 70% ethanol using a 1000µl wide-bore tip.
Transfer to a new 1.5 ml tube.
Ethanol 70% [Note: freshly prepared]Contributed by users
1.5 mL Eppendorf tubesContributed by users
Centrifuge for 10 min at 5,000g at 4°C to pellet DNA. set centrifuge deceleration speed to level3
5000 x g, 4°C, 00:10:00 , Acc 9 / Dec 3
10m
Remove as much supernatant as possible, avoiding touching the pellet.
Air dry the pellet 10 min at RT.
00:10:00 Room temperature
10m
Resuspend the pellet with 50 µL of TE 1x prewarm to 50°C
Tris-EDTA (TE) buffer pH 8.0 1XContributed by users
Incubate for 1hour at 50°C, then overnight at RT.
01:00:00 50 °C
Overnight Room temperature
1h
Sample QC
Sample QC
Quantify your sample with a Qubit HS.
Analyse 1 µL in a UV spectrophotometer (e.g. Nanodrop).
Visualise 1 µL of sample to estimate the molecular weight. (Tapestation)
Results
Results
QC results of extraction of 4 individuals
| Weight mg | [c] ng/µl | Nanodrop A260/A280 | Nanodrop A260/A230 | Tapestation kb | |
| 63 | 39.4 | 1.82 | 1.86 | 57 | |
| 40 | 30.9 | 1.88 | 2.20 | 57 | |
| 65 | 82.1 | 1.78 | 1.82 | 50 | |
| 43 | 197 | 1.86 | 2.33 | 26 |
Tapestation profiles