May 22, 2024

HTTM : gDNA extraction V.3

DOI
https://dx.doi.org/10.17504/protocols.io.q26g7yzz3gwz/v3
  • 1Université de Sherbrooke
Antoine Champie
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g7yzz3gwz/v3
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: May 21, 2024
Last Modified: May 22, 2024
Protocol  Integer ID: 100192
Keywords: gdna extraction part of the httm protocol, gdna extraction part, extraction of gdna, gdna, mutated cell pellet, httm protocol, part of the httm protocol, cell pellet, transposon, extraction
Abstract
Part of the HTTM protocol dedicated to the extraction of gDNA from transposon mutated cell pellets.
Attachments
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Materials
  • Homemade DNA lysis Buffer :
AB
ComponentAmount 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 :
AB
ComponentAmount 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)
AB
ComponentAmount for 1000ml of solution
Water960ml
NaOH40g
Triton X-1001,5ml
Mix well and store in a base resistant container.

  • HCl 1.5N + Triton X100 0,15% (v/v)
AB
ComponentAmount for 1000ml of solution
Water873,5ml
HCl Stock (37%)125ml
Triton X-1001,5ml
Mix well and store in an acid resistant container.
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)
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.