eDNA extraction: phenol-chloroform-isoamyl alcohol DNA purification from filters stored in Longmire buffer
Ana Ramón-Laca, Abigail Wells, Linda Park
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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.
Steps
DNA extraction
heat-shock Filters in 2 ml of Longmire buffer at 95C for 5' and then allow samples to cool to room temperature
digestion add 100 µl proteinase K (final concentration 1 mg ml-1)
incubation 56C at 120 rpm for 2h
phase lock set up add ca. 800 µl of vacuum grease with a syringe onto the wall of the tube of the 5 ml tube
PCI (25:24:1) add 2 ml of phenol-chloroform-isoamyl (25:24:1) pH 8
centrifugation shake well and spin 13.3 x g for 5' at 4C
phase lock set up place ca. 800 µl of vacuum grease in 2 sets of empty tubes for CI. (These can be prefilled for convenience)
CI add 2.2 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.
centrifugation shake and spin 13.3 x g for 5' at 4C
CI add 2.2 ml of chloroform:isoamyl 24:1 and decant aqueous layer from step 9
centrifugation shake and spin 13.3 x g for 5' at 4C
Isopropanol add 2 ml of isopropanol (can be prefilled for convenience), add 80 µl of 5M NaCl and decant aqueous layer from step 11
mixing invert several times
precipitation overnight (or 2 h) at room temperature
centrifugation spin 13.3 x g for 30' at 4C
wash x2 pour liquid off slowly and add 800 µl of ice cold 70% EtOH
centrifugation shake and spin 13.3 x g for 5' at 4C and repeat wash
drying pour liquid off slowly and allow tubes to dry for 1 h or until dry
resuspension once they are dry, resuspend in 100 µl TE buffer (warm - 37C)
storage store in the freezer (-20-80 C)
