Dec 12, 2023
Version 3
Nucleic acids extraction from single cell using MasterPure Complete DNA purification (Epicenter) V.3
- 1UMR7144-Roscoff Marine Station
Protocol Citation: Sarah Romac 2023. Nucleic acids extraction from single cell using MasterPure Complete DNA purification (Epicenter). protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9mk3qg3e/v3Version created by Sarah Romac
Manuscript citation:
Biard et al. Protist 2015. http://dx.doi.org/10.1016/j.protis.2015.05.002
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: December 12, 2023
Last Modified: December 12, 2023
Protocol Integer ID: 92159
Abstract
Radiolaria are protists which can't be cultivated. These microoganisms have to be isolated by single-cell for genetic identification and it can be difficult to get clean DNA.
Here we optimized a DNA extraction protocol from protist single-cell.
It works very well on single-cell Radiolaria, Foraminifers, but also Ciliates, Dinoflagelletes, Diatoms.
Materials
Material and Equipment :
- Glass bent micropipettes.
- Silicone Tubing (Cole-Parmer, Tube silicone platinium LS T17 ref. FV-96410-17
- StereoMicroscope or binocular microscope.
- Petridishes Diameter 70mm.
- Centrifuge 5417R (Eppendorf)
- Thermomixer Eppendorf
Kit, Reagents and Chemicals :
- MasterPure Complete DNA and RNA purification kit (Epicenter Illumina, ref MC85200)
Enzymes (Proteinase K, RNase, DNase) are stored at -20°C
Other reagents from the kit are stored at room temperature.
- Ethanol absolute, molecular biogy quality.
- Propan-2-ol quality molecular biology.
- 0,22µm filtered Seawater or artificial Seawater.
- Ice for enzyme storage during labwork.
Safety warnings
Wear labcoat, gloves.
Decontaminate all the surface area, and equipments (rotor, racks, pipettes...) with Ethanol 70% and DNA away.
Works with filter tips.
Before start
Prepare Ethanol 70% from absolute Ethanol : Mix 35 mL of absolute Ethanol with 15 mL nuclease-free water in a Falcon 50mL Store at -20°C.
1. Cell Isolation
1. Cell Isolation
Isolate individually protist cells (at least 50µm in length) using a glass bent micropipette under a binocular microscope.
Wash each cell in three successive baths of 0.22µm-filtered and sterile seawater.
Transfer subsequently cells in a 1.5mL sterile microtube.
Add 30 μL of lysis buffer (Tissue and Cell Lysis Solution from MasterPureTM DNA and RNA Purification Kit, Epicenter) and store at -20°C.
2. Cell lysis
2. Cell lysis
Pellet cells by centrifugation (2 min at Vmax), throw the supernatant, let ~25-30 µL of liquid.
Dilute 1 µL of Proteinase K in 300 µL de lysis solution Tissue et Cellule for each sample. Vortex 10 sec for resuspending cells (facultative).
Add 300 µL of mix Proteinase K + lysis solution Tissue et Cellule in each sample. Vortex.
Incubate 15 min at 65°C , 1000 rpm. Put samples in ice 3-5 min.
3. Total nucleic acids precipitation
3. Total nucleic acids precipitation
Add 150 µL MPC reagent to 300 µL of lysed sample. Vortex.
Spin 10 min at 11 000 g, 4°C. If there is no pellet, add more 25 µL MPC buffer and spin again 10 min at 11 000 g, 4°C.
Transfer the supernatant ion a new clean microtube (1,5 mL), discard the pellet.
(To keep the squeletton, keep the tube with the pellet, add 500 µL MilliQ Water and store at -20°C).
Add 500 µL of Isopropanol. Mix per inversion. Spin 10min at Vmax, 4°C.
Discard the supernatant with precaution, without touching the pellet.
Add 500 µL of Ethanol 70%. Don’t vortex, mix gently the support. Spin 5min at Vmax ;at 4°C.
Discard a maximum of supernatant with precaution, without touching the pellet.
Let dry 5-10 min at room temperature. The pellet should become transparent.
Elute in 25 µL of TE1x buffer.
Vortex and spin shortly.
Store at - 80°C.