May 22, 2024
Version 3
HTTM : gDNA extraction V.3
- Antoine Champie1,
- Amélie De Grandmaison1,
- Sebastien Rodrigue1
- 1Université de Sherbrooke
External link: https://doi.org/10.1371/journal.pone.0283990
Protocol Citation: Antoine Champie, Amélie De Grandmaison, Sebastien Rodrigue 2024. HTTM : gDNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7yzz3gwz/v3Version created by Antoine Champie
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: August 24, 2022
Last Modified: May 22, 2024
Protocol Integer ID: 100192
Abstract
Part of the HTTM protocol dedicated to the extraction of gDNA from transposon mutated cell pellets.
Attachments
Image Attribution
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Materials
- Homemade DNA lysis Buffer :
| A | B | |
| Component | Amount for 1000ml of solution | |
| CTAB 2% | 20g | |
| 1,5M Guanidine HCl | 143,2g | |
| 10mM Tris HCl | 1,57g |
Mix well and adjust pH to 8.0.
- Homemade wash solution :
| A | B | |
| Component | Amount for 1000ml of solution | |
| Ethanol 100% | 800ml | |
| Tris HCl 1M pH 8,0 | 10ml | |
| NaCl 4M | 25ml | |
| EDTA 0,5M | 2ml |
Mix well and adjust pH to 8.0.
- Elution Buffer (Low TE Buffer): 10 mM Tris-HCl (pH 8.0) + 0.1 mM EDTA
Solutions for plate regeneration, from this protocol : (1)https://doi.org/10.1016/j.ab.2008.10.021.
- NaOH 1N + Triton X100 0,15% (v/v)
| A | B | |
| Component | Amount for 1000ml of solution | |
| Water | 960ml | |
| NaOH | 40g | |
| Triton X-100 | 1,5ml |
Mix well and store in a base resistant container.
- HCl 1.5N + Triton X100 0,15% (v/v)
| A | B | |
| Component | Amount for 1000ml of solution | |
| Water | 873,5ml | |
| HCl Stock (37%) | 125ml | |
| Triton X-100 | 1,5ml |
Mix well and store in an acid resistant container.
DNA extraction
DNA extraction
2h 5m
Prepare the lysis solution by adding 165 µL of proteinase K to 66 mL of homemade lysis buffer and mix well.
Add 600 µL of lysis solution to each well of the deep-well plate and resuspend the pellet.
Cover with an adhesive aluminum foil and incubate at 55 °C for 01:00:00 .
1h
While still warm, add 260 µL of ethanol 100%, without overmixing.
Note
Overmixing will result in DNA agglomeration and difficulty with the extraction.
Transfer immediately to a deep-well plate fitted with an array of silica columns.
Centrifuge twice at 3270 x g, 00:10:00 .
10m
Discard flowthrough and add 500 µL of wash solution.
Centrifuge at 3000 x g, 00:10:00
10m
Repeat steps 7 and 8.
Discard flowthrough.
Centrifuge at 3000 x g, 00:05:00 to eliminate traces of wash solution.
5m
Discard flowthrough.
Add a collector plate between the silica column array and the deep-well plate.
Add 50 µL of low TE to the silica matrix in each well.
Cover with an adhesive aluminum foil and incubate at55 °C for 00:15:00 .
15m
Centrifuge at 3270 x g, 00:05:00 .
5m
Silica array regeneration (Optional)
Silica array regeneration (Optional)
1h 5m
Put the contaminated silica array on an empty deep-well plate.
Add 150 µL of 1N NaOH + 0.15%(v/v) Triton X-100 to each well.
Incubate at Room temperature for 00:05:00
5m
Centrifuge 3000 x g, 00:02:00
2m
Add 200 µL of 1,5N HCl+ 0,15% (v/v) Triton X-100 to each well.
Incubate at Room temperature for 00:30:00
30m
Centrifuge 3000 x g, 00:02:00
2m
Add 150 µL of 1N NaOH + 0,15%(v/v) Triton X-100 to each well.
Incubate at Room temperature for 00:05:00
5m
Centrifuge 3000 x g, 00:02:00
2m
Collect the flowthrough in a beaker. Neutralize pH if needed and dispose of the flow through.
Add 600 µL of ddH2O to each well.
Centrifuge 3000 x g, 00:05:00
5m
Silica columns array are ready to be reused.