Sep 09, 2021

Public workspaceeDNA extraction: phenol-chloroform-isoamyl alcohol DNA purification from filters stored in Longmire buffer

 Forked from eDNA extraction: phenol-chloroform-isoamyl alcohol DNA purification from filters stored in Longmire buffer
PLOS One
DOI
dx.doi.org/10.17504/protocols.io.bx4ypqxw
  • Abigail Wells1,
  • Linda Park1
  • 1NWFSC-NOAA
Ana Ramón-Laca
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DOI: dx.doi.org/10.17504/protocols.io.bx4ypqxw
External link: https://doi.org/10.1371/journal.pone.0257773
Protocol Citation: Abigail Wells, Linda Park 2021. eDNA extraction: phenol-chloroform-isoamyl alcohol DNA purification from filters stored in Longmire buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.bx4ypqxw
Manuscript citation:
Ramón-Laca A, Wells A, Park L (2021) A workflow for the relative quantification of multiple fish species from oceanic water samples using environmental DNA (eDNA) to support large-scale fishery surveys. PLoS ONE 16(9): e0257773. doi: 10.1371/journal.pone.0257773
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: September 09, 2021
Last Modified: September 09, 2021
Protocol Integer ID: 53112
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Abstract
This is an organic DNA extraction method for filters preserved in 2 ml of Longmire buffer that uses a phase lock to allow easy decanting of the aqueous layer instead of pipetting.
Guidelines
To make TlowE:
To make 50 ml
1. 500 µl of 1 M Tris-HCL, pH 8.0
2. 10 µl of 0.5 M EDTA, pH8.0
3. up to 50 ml of nuclease free H2O
Materials
  •  proteinase k 20 mg ml-1
  •  Dow Corning high vacuum grease 5.3 oz
  •  Phenol-chloroform-isoamyl (25:24:1)
  •  Chloroform:isoamyl 24:1
  •  Isopropyl
  •  Ethanol (95%)
  •  5 ml LoBind tubes Eppendorf
  •  Pipettes and tips (5 ml, 1 ml, 200 µl)
  •  1.5 ml LoBind tubes Eppendorf
  •  Zymo One-Step inhibitor removal plates
  •  Incubator
  •  Centrifuge with a rotor with capacity for 5 ml tubes
  •  Tube racks (5 ml, 2 ml)
  •  Tube squeezing tool and sterile 15 ml syringes with wings or rings for grease dispensing
  •  Laboratory chemical fume hood or biosafety cabinet
  •  5 M NaCl
  •  1 M Tris-HCL, pH 8.0
  • 0.5 M EDTA, pH8.0
  • H2O
Safety warnings
Phenol and Chloroform are very corrosive and irritant chemicals. Always perform these extractions in fume hoods, wear a lab coat, safety glasses, nitrile gloves and any other PPE. Also, always use resistant plasticware. Please refer to Safety Datasheets for more information. Phenol is volatile and can burn your skin and damage your eyes. Chloroform can make you faint and can even be lethal
DNA extraction
DNA extraction
2h 50m
2h 50m
heat-shock Filters in Amount2 mL of at Temperature95 °C forDuration00:05:00 min and then allow samples to cool to room temperature
Protocol
Longmire lysis buffer
NAME

Longmire lysis buffer

CREATED BY
Ana Ramón-Laca



5m
975 ml double-distilled water
100 ml of 1 M Tris-HCL, pH 8.0
200 ml of 0.5 M EDTA, pH8.0
2 ml of 5 M NaCl
25 ml of 20% SDS (w/v) Filter the buffer with an autofill PES bottle top filtration device (sterile 500ml, 0.22 µm)
digestion add Amount100 µL proteinase K (final concentration Concentration1 mg/mL )

incubation Temperature56 °C at Shaker120 rpm for Duration02:00:00

2h
phase lock set up add ca. Amount800 µL of vacuum grease with a syringe onto the wall of the 5ml tube without touching the lysate to avoid cross-contamination
Equipment
Eppendorf 5415R Refrigerated Centrifuge
NAME
Refrigerated Centrifuge
TYPE
Eppendorf
BRAND
EP-5415R
SKU


add 2 ml of phenol-chloroform-isoamyl (25:24:1) Ph8
pH is critical for good results
Critical
Toxic
shake well and centrifuge Centrifigation13300 x g, 4°C, 00:05:00

5m
place ca. Amount800 µL of vacuum grease in 2 sets of empty tubes for CI. (These can be prefilled for convenience)

add Amount2.2 mL of chloroform:isoamyl 24:1 and decant aqueous layer from step 6.
Important!! only add the chloroform just before use (while the tubes with PCI are in the centrifuge) so it does not affect the phase lock if grease is at the bottom.

Video

Critical
shake well and centrifuge Centrifigation13300 x g, 4°C, 00:05:00
5m
Go togo to step #8

add Amount2 mL of isopropanol (can be prefilled for convenience), add Amount80 µL of Concentration5 Molarity (M) NaCl and decant aqueous layer from step 11

invert several times
precipitation overnight (or 2 h) at room temperature
centrifuge Centrifigation13300 x g, 4°C, 00:30:00
30m
pour liquid off slowly and add Amount800 µL of ice cold Concentration70 % (v/v) EtOH

shake well and centrifuge Centrifigation13300 x g, 4°C, 00:05:00 and Go togo to step #15

5m
pour liquid off slowly and allow tubes to dry for 1 h or until dry
once they are dry, resuspend in Amount100 µL TE buffer (warm Temperature37 °C to favor elution)

store in the freezer (Temperature-20-80 °C )