HTTM : DNA Extraction
Antoine Champie, Amélie De Grandmaison
Abstract
Part two of the HTTM protocol. A low-cost and high-throughput Tn-seq protocol.
This part cover the DNA extraction from cell pellets of transposon insertion mutants and subsequent silica columns regeneration.
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 cover 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 4000x 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 3000x 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 200µLof ddH2O to each well.
Centrifuge 3000x g.
Repeat steps 25 and 26.
Silica columns array are ready to be reused.
