Nov 16, 2022
Version 1
DNA Extraction of Placenta Tissue V.1
This protocol is a draft, published without a DOI.
- 1University of California, San Diego
Protocol Citation: Cayla Mason 2022. DNA Extraction of Placenta Tissue . protocols.io https://protocols.io/view/dna-extraction-of-placenta-tissue-cjejujcn
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: November 16, 2022
Last Modified: November 16, 2022
Protocol Integer ID: 72875
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Abstract
This protocol describes extracting DNA from placenta tissue, starting with dissociation.
Materials
1.0 mm diameter zirconia/silica beads, BioSpec Products, catalog 11079110z
DNeasy Blood and Tissue Kit, Qiagen, catalog 69504
100% ethanol
Nuclease-free water
2 mL screw-cap tubes with O-ring gaskets
Nuclease-free low-retention 1.5 mL microcentrifuge tubes
Tissue Dissociation and Lysis
Tissue Dissociation and Lysis
2h
2h
Place placenta tissue samples in screw cap tubes with O-ring gaskets on ice to thaw.
Label a 1.5 mL microcentrifuge tube for each placenta sample. Aliquot 1 mL silica beads per tube.
Add 540 uL Buffer ATL and 20 uL Proteinase K to each tissue. Pour in prepared beads.
Tightly close tubes. Place tubes in Bead Beater, and tightly close screw-on parts. Homogenize for 2 minutes.
Incubate lysates with beads in 56C water bath or heat block for 2 minutes.
Transfer lysate to pre-labeled microcentrifuge tubes. Do not discard beads.
Add 600 uL Buffer AL to screw-cap tubes with beads. Vortex and quick spin. Transfer supernatant to microcentrifuge tube with lysate. Discard screw-cap tube with beads.
Vortex lysate until homogenized.
Incubate lysates 56C for 10 minutes. Quick spin.
Freeze at -80C or proceed with DNA extraction.
DNA Extraction
DNA Extraction
1h 30m
1h 30m
Thaw lysates at room temperature.
Prepare columns and buffers. Add 25 mL 100% ethanol to Buffer AW1 and 30 mL 100% ethanol to Buffer AW2.
Add one-half volume (approximately 500 uL) 100% ethanol to lysate. Mix thoroughly by vortexing.
Pipet maximum 700 uL of the mixture to column. Centrifuge at >=6000g for 1 minute. Discard flow through. Repeat this step until all lysate has passed through column.
Add 500 uL Buffer AW1. Centrifuge for 1 minute at >=6000g. Discard flow-through and collection tube.
Place the spin column in a new 2 mL collection tube. Add 500 uL Buffer AW2. Centrifuge for 3 minutes at 20000g. Discard flow-through and collection tube.
While spinning, label 1.5 mL microcentrifuge tubes for elution.
Transfer column to pre-labeled microcentrifuge tubes.
Elute DNA by adding 100 uL Buffer AE or nuclease-free water to the center of the membrane. Incubate for 1 minute at room temperature. Centrifuge for 1 minute at >=6000g.
Repeat step 18.
Store eluted DNA at -20C.