Nucleic acids extraction from single cell using MasterPure Complete DNA purification (Epicenter)
Sarah Romac
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.
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.
Steps
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
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
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.
