HTTM : gDNA extraction
Antoine Champie, Amélie De Grandmaison, Sebastien Rodrigue
Abstract
Part of the HTTM protocol dedicated to the extraction of gDNA from transposon mutated cell pellets.
Attachments
HDTM _ Protocol-2.pdf
Steps
DNA extraction
Prepare the lysis solution by adding 165µL of proteinase K to 66mL of homemade lysis buffer and mix well.
Add 600µLof 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 1h 0m 0s.
While still warm, add 260µLof ethanol 100%, without overmixing.
Transfer immediately to a deep-well plate fitted with an array of silica columns.
Centrifuge twice at 3270x g .
Discard flowthrough and add 500µL of wash solution.
Centrifuge at 3000x g
Repeat steps 7 and 8.
Discard flowthrough.
Centrifuge at 3000x g to eliminate traces of wash solution.
Discard flowthrough.
Add a collector plate between the silica column array and the deep-well plate.
Add 50µLof low TE to the silica matrix in each well.
Cover with an adhesive aluminum foil and incubate at55°C for 0h 15m 0s.
Centrifuge at 3270x g .
Silica array regeneration (Optional)
Put the contaminated silica array on an empty deep-well plate.
Add 150µL of 1N NaOH + 0.15%(v/v) Triton X-100 to each well.
Incubate at Room temperature for 0h 5m 0s
Centrifuge 3000x g
Add 200µL of 1,5N HCl+ 0,15% (v/v) Triton X-100 to each well.
Incubate at Room temperature for 0h 30m 0s
Centrifuge 3000x g
Add 150µL of 1N NaOH + 0,15%(v/v) Triton X-100 to each well.
Incubate at Room temperature for 0h 5m 0s
Centrifuge 3000x g
Collect the flowthrough in a beaker. Neutralize pH if needed and dispose of the flow through.
Add 600µLof ddH2O to each well.
Centrifuge 3000x g
Silica columns array are ready to be reused.