Chlamydomonas

RNA Extraction (added by Leonardo Magneschi)

1) Pellet cells, remove surnatant and freeze them in liquid nitrogen (store at -80°C)

2) Resuspend pellet in 750 µl of SDS-EB* buffer + 75 µl Sodium Acetate 3M pH 5

3) Add 750 µl phenol:chloroform (1:1), vortex and centrifuge 5 minutes at 12.000 g to separate phases

4) Transfer aqueous phase to a new tube

5) Repeat step 3-4 two more times

6) Extract with chloroform, vortex and centrifuge 5 minutes at 12.000 g

7) Transfer aqueous phase to a new tube

8) Add an equal volume of LiCl 8M and incubate minimum 4 hours at 4°C

9) Centrifuge 20 minutes at 13.000 g, 4°C

10) Wash pellet with 70% ethanol

11) Dry pellet and resuspend in RNase-free water

12) Assay quality of RNA by 1% agarose gel electrophoresis and read absorbances at 260/280 nm

13) Do Dnase treatment

* SDS-EB buffer: 2% SDS, 400 mM NaCl, 40 mM EDTA, 100 mM Tris-HCl (pH=8)

Genomic DNA Extraction (added by Leonardo Magneschi)

1) Pellet cells, remove surnatant and freeze them in liquid nitrogen (store at -80°C)

2) Resuspend pellet in 750 µl of SDS-EB* buffer:water (1:1)

3) Add 750 µl phenol:chloroform (1:1), vortex and centrifuge 5 minutes at 12.000 g to separate phases

4) Transfer aqueous phase to a new tube

5) Add 2 to 5 µl of RNase and incubate 30 minutes at 37°C

6) Repeat step 3 two more times

7) Extract with chloroform, vortex and centrifuge 5 minutes at 12.000 g

8) Transfer aqueous phase to a new tube

9) Add two volumes of 100% ethanol and incubate 1 hour at -20°C

10) Centrifuge 20 minutes at 13.000g, 4°C

11) Wash the pellet once with with 70% ethanol

12) Dry pellet and resuspend in ultrapure water (depending on the pellet, 50-100 µl)

* SDS-EB buffer: 2% SDS, 400 mM NaCl, 40 mM EDTA, 100 mM Tris-HCl (pH=8)