Apr 08, 2024
Inexpensive DNA Extraction Protocol
This protocol is a draft, published without a DOI.
- Valerie Warhol1
- 1Carnegie Museum of Natural History
Protocol Citation: Valerie Warhol 2024. Inexpensive DNA Extraction Protocol. protocols.io https://protocols.io/view/inexpensive-dna-extraction-protocol-dbtt2nnn
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: April 07, 2024
Last Modified: April 08, 2024
Protocol Integer ID: 97875
Keywords: dna extraction protocol, inexpensive dna extraction protocol, dna extraction, extraction of dna, extraction protocol, dna, using inexpensive reagent, inexpensive reagent, extraction, doing extraction, protocol, fungus sample, enzyme
Abstract
This is a protocol for doing extraction of DNA using inexpensive reagents rather than enzymes or kits. It can be used for plant, animal, or fungus samples.
Materials
Forceps (for handling sample)
Pestle (for grinding sample in microtube)
Hinged 1.5 µL microtubes (3 per sample)
Microcentrifuge
Water bath
Adjustable micropipette 1-10 μL
Adjustable micropipette 10-100 μL
Adjustable micropipette 100-1000 μL
Pipette tips
Molecular grade water (100 µL per sample)
Silica resin (silica dioxide 50% w/v in water) (3 µL per sample)
Wash buffer (diluted 1:1 in 95% ethanol) (1 mL per sample)
Guanidine Hydrochloride 6M (250 µL per sample)
Protocol materials
Guanidine Hydrochloride 6MCarolina Biological SupplyCatalog #C33427
Silica ResinCarolina Biological SupplyCatalog #C33426
Wash BufferCarolina Biological SupplyCatalog #C33428
Prepare sample and equipment
Make sure all instruments, such as forceps and pestle, are clean and sterile. Make sure Wash BufferCarolina Biological SupplyCatalog #C33428 has been diluted 1:1 in 95% ethanol.
Prepare water bath at 65 °C .
Dissect sample from specimen. (This will be a piece of tissue approximately 10–20 mg. For small arthropods such as beetles or spiders, 1 or 2 legs will typically suffice.) Return specimen to freezer.
If sample was stored in ethanol, let sample dry for 5–10 minutes.
Prepare a clean 1.5 mL hinged tube by writing sample ID on it and filling with 250 µL of Guanidine Hydrochloride 6MCarolina Biological SupplyCatalog #C33427
Lyse cells
11m
Put sample in tube. Grind sample with pestle until broken up into tiny pieces.
Incubate sample tube in 65 °C water bath for 00:10:00 .
10m
Remove tube and lower temperature of water bath to 57 °C .
Centrifuge tube for 00:01:00 at maximum speed to pellet debris.
1m
Remove Silica ResinCarolina Biological SupplyCatalog #C33426 from refrigerator.
Label a clean 1.5 µL tube with sample number. Transfer 150 µL of the supernatant to the clean tube. Discard old tube containing debris.
Bind DNA
5m 30s
Add 3 µL of Silica ResinCarolina Biological SupplyCatalog #C33426 to tube. Mix well by pipetting up and down several times.
Close tube and incubate for 00:05:00 in 57 °C water bath.
5m
Centrifuge for 00:00:30 at maximum speed to pellet the resin.
30s
Use a pipette with a fresh tip to remove the supernatant, being careful not to disrupt the pellet.
Wash
1m
Remove molecular grade water from refrigerator and Wash BufferCarolina Biological SupplyCatalog #C33428 from freezer.
Add 500 µL of ice-cold Wash BufferCarolina Biological SupplyCatalog #C33428 to the pellet. Mix well by pipetting up and down several times to resuspend the silica resin.
Close the tube and centrifuge for 00:00:30 at maximum speed to pellet the resin.
30s
Use a pipette with a fresh tip to remove the supernatant, being careful not to disrupt the pellet.
Again, add 500 µL of ice-cold Wash BufferCarolina Biological SupplyCatalog #C33428 to the pellet. Mix well by pipetting up and down to resuspend the silica resin.
Close the tube and centrifuge for 00:00:30 at maximum speed to pellet the resin.
30s
Return wash buffer to freezer.
Use a pipette with a fresh tip to remove the supernatant, being careful not to disrupt the pellet. Spin the tube briefly to collect any remaining drops of supernatant, and then remove these with a pipette.
Elute DNA
30s
Add 100 µL of molecule grade water to the silica resin and mix by pipetting up and down several times.
Incubate the mixture at 57 °C for 5 minutes.
Centrifuge for 00:00:30 at maximum speed to pellet the resin.
30s
Label a clean 1.5 µL tube with sample number. Transfer 90 µL of the supernatant to the clean tube, being careful not to disturb the pellet. Discard old tube containing the resin.
Store sample in freezer until ready to PCR. If going directly to PCR, put sample in refrigerator.