Nov 01, 2022
Foreskin Tissue DNA Extraction
- 1Center for Global Infectious Disease Research, Seattle Children's Research Institute;
- 2Division of Infections Disease, Department of Pediatrics, University of Washington School of Medicine
Protocol Citation: Brandon Maust 2022. Foreskin Tissue DNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2774jg1y/v1
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: October 31, 2022
Last Modified: November 01, 2022
Protocol Integer ID: 72091
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Abstract
Extracts total DNA from foreskin tissue, as used in Maust et al. 2022
Materials
Supplies
2 ml screw top microcentrifuge tubes
3mm steel beads (1 per sample)
scalpels
plate sealing film
proteinase K (20 mg/ml)
Absolute ethanol
PowerSoil Pro plates (Cat no 19311)
DNeasy 96 Powersoil Pro QIAcube HT kit (Cat no 47021)
Equipment
scale
heat block
Qiagen TissueLyser II
Qiagen QIAcube HT
Preparation
Preparation
Add steel bead to 1.5 ml tube
Add 800 µL CD1 to tube
Weigh tube (with bead and solution)
Tissue
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
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
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