Foreskin Tissue DNA Extraction
Brandon Maust
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Abstract
Extracts total DNA from foreskin tissue, as used in Maust et al. 2022
Steps
Preparation
Add steel bead to 1.5 ml tube
Add 800 µL CD1 to tube
Weigh tube (with bead and solution)
Tissue
Dissect approximately 25 mg (3 mm3) piece of tissue
Fragment tissue with scalpel
Add tissue to tube (with bead and solution)
Weigh tube and calculate net tissue weight.
Pre-robot processing
Process tubes on TissueLyzer II, 2 min at 30 Hz
Rotate block 180º and repeat
Centrifuge tubes briefly to collapse foam
Spin PowerBead Pro plate to ensure beads are settled at the bottom
Add contents of each specimen tube (except steel bead) to a PowerBead plate well
Add 5 µL of 20 mg/ml proteinase K to each well
Seal plate with film
Vortex briefly to mix
Incubate plate at 65º C for 60 min or until tissue is mostly digested
Process plate on TissueLyser II, 5 min at 25 Hz
Rotate plate 180º and repeat
Centrifuge plate at 3000 x g for 7.5 min
Transfer approximately 350 µL supernatant from each well to fresh S-block using well-vator
add 300 µL of solution CD2 to each well and mix thoroughly by pipetting
Seal the plate with film
Centrifuge at 3000 x g for 7.5 min at room temperature
Avoiding the pellet, transfer 500 µL of supernatant to fresh S-block using well-vator
QIAcube
Follow DNeasy 96 PowerSoil Pro Protocol for QIAcube HT (page 17 onward)
Elute in maximum volume (120 µL)
Incude vacuum performance check
Seal plate and freeze at -20ºC
