May 02, 2018
Version 2
Phenol/chloroform extraction of DNA from cyanobacteria V.2
- Robbie Martin1,
- Steven W. Wilhelm1
- 1University of Tennessee, Knoxville
- The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Protocol Citation: Robbie Martin, Steven W. Wilhelm 2018. Phenol/chloroform extraction of DNA from cyanobacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.ptndnme
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 27, 2018
Last Modified: May 02, 2018
Protocol Integer ID: 11854
Keywords: DNA extraction, cyanobacteria
Abstract
Phenol/chloroform-based DNA extraction from cells pre-treated with RNase A, lysozyme, proteinase K, and SDS. The protocol was optimized for extracting DNA from Microcystis aeruginosa, but works well for other cyanobacteria.
Materials
MATERIALS
Sodium acetateP212121
RNase AQiagenCatalog #19101
Chloroform/IAAAmrescoCatalog #X205
TE buffer
Phenol
70% Ethanol
Proteinase KThermo Fisher ScientificCatalog #EO0491
Sodium Dodecyl Sulfate, 100gmPromegaCatalog #H5113
LysozymeBio Basic Inc.Catalog #LDB0308.SIZE.1g
100% Ethanol
Safety warnings
Pre-treatment of cells with RNase A, lysozyme, proteinase K, and SDS
Pre-treatment of cells with RNase A, lysozyme, proteinase K, and SDS
Concentrate ~4 mL of cellular culture in an Eppendorf tube by centrifugation at 10,000 xg for ~5 min.
4 mL cellular culture
00:05:00 centrifugation at 10,000 xg
Discard supernatant.
Resuspend pellet in 425 µL of standard TE buffer.
425 µL TE buffer
Note
Standard TE buffer
-10 mM Tris
-1 mM EDTA
-pH 8
Add 50 µL of 100 µg/mL RNase A TER buffer (RNase A plus TE buffer) for a final concentration of 10 µg/mL RNase A.
50 µL RNase A TER buffer
Add 25 µL of 100 mg/mL lysozyme for a final concentration of 5 mg/mL lysozyme.
25 µL of 100 mg/mL lysozyme
Incubate at 37 °C for 20 minutes.
37 °C incubation
00:20:00 incubation
Add 50 µL of 1 mg/mL proteinase K for a final concentration of 100 µg/mL proteinase K.
Add 50 µL of 10% sodium dodecyl sulfate.
50 µL 10% sodium dodecyl sulfate
Incubate at 50 °C for 2 hr.
50 °C incubation
02:00:00 incubation
Phenol/chloroform extraction
Phenol/chloroform extraction
Add 250 mL phenol (pH 8.0). Mix gently and completely.
250 mL phenol (pH 8.0)
Add 250 mL chloroform/isoamyl alcohol (24:1), mix gently and completely.
250 mL chloroform/isoamyl alcohol (24:1)
Spin at maximum speed in benchtop centrifuge for at least 2 minutes.
Note
Longer spin times, (up to 10 min.) may help in separating the phases.
Transfer aqueous (top) layer to clean Eppendorf tube without removing any of the organic layer.
Remove the last of the aqueous layer along with some of the organic layer (to ensure all aqueous volume is collected) and add it to a new, clean Eppendorf tube for later re-extraction.
Note
This re-extraction will significantly increase DNA recovery.
- Repeat steps 10, 11 and 12 twice (a total of 3 phenol extractions) or until no visible protein layer is seen.
- Each time, remove the last of the aqueous layer along with some of the organic layer and add to the 're-extraction' Eppendorf tube (refer to step 14).
At least 3 phenol extractions, or until no visible protein layer is seen.
Note
Protein layer will be white scum at interface.
Add to 're-extraction' Eppendorf tube.
Add 500 µL of chloroform and mix gently.(1/2)
500 µL chloroform
Spin at maximum speed in benchtop centrifuge for at least 2 minutes. (1/2)
Note
This will help remove traces of phenol.
00:02:00 centrifugation
Transfer aqueous (top) layer to clean Eppendorf tube without removing any of the organic layer.(1/2)
Repeat steps 16-18 to remove all phenol. (2/2)
Repeat phenol removal
Extract the mixed aqueous/organic contents of the 're-extraction' Eppendorf tube and follow extraction steps 10-19. Combine the final aqueous layer with that collected above.
extraction steps for aqueous/organic contents
Ethanol precipitation
Ethanol precipitation
Add 0.1 volume of 3 M sodium acetate to the collected aqueous phase and mix gently.
Add 2 volumes of ice cold 100% ethanol and gently mix well.
Note
Strings of precipitating DNA should become visible.
Place tube in -80 °C freezer until ethanol mixture is partially frozen (~1 hr.)
-80 °C freezer
01:00:00 approximate time for partial ethanol freeze
Spin at maximum speed in benchtop centrifuge at 4 °C for 30 min.
00:30:00 centrifugation
4 °C centrifugation
Discard supernatant and very carefully aspirate the remaining droplets of liquid from the tube without disrupting the DNA pellet.
Wash the DNA pellet by adding 500 µL of ice-cold 70% ethanol to the tube and pipetting it gently, several times.
500 µL ice-cold 70% ethanol
Spin at maximum speed in a benchtop centrifuge at 4 °C for 15 min.
00:15:00 centrifugation
4 °C centrifugation
Discard supernatant and carefully aspirate the remaining droplets of liquid from the tube without disrupting the DNA pellet.
Place in a heater block at ~37 °C for less than 5 minutes to evaporate all ethanol.
Note
Be careful! DO NOT dry to completion! Alternatively, you can air dry to remove the ethanol.
00:04:59 ethanol drying
37 °C heater block
Re-suspend the DNA pellet in 50-100 µL of TE buffer or water (depending on downstream use and needed concentration).
50 µL TE buffer or water
Freeze at -20 or -80 °C.