Preparation of yeast total RNA
Good quality total RNA can be extracted from yeast cells.
Operation method
Preparation of yeast total RNA
Materials and Instruments
AE Buffer. Phenol 10% SDS Phenol Chloroform Anhydrous ethanol Ethanol . 3mol L sodium acetate Water without RNase Move I. Materials and equipment Caveat Replacing AE equilibrated phenol with Tris-Cl equilibrated phenol at PH 8. 0 also gives good results. For more product details, please visit Aladdin Scientific website.
High speed centrifuge
1) AE buffer: 50 mmol/L sodium acetate (PH5.3), lOmmol/LEDTA.
2) Phenol (pre-equilibrated with AE buffer).
3)10% SDS
4) Phenol : Chloroform (1:1).
5) Chloroform
6) Anhydrous ethanol and 80% ethanol.
7) 3mol/L sodium acetate
8) RNase-free water
9) high speed centrifuge.
II. Methods of operation
1) Collect l0 ml of yeast cell culture by centrifugation and resuspend with 40ul of AE buffer. Transfer to a 1.5 ml centrifuge tube.
2) Add 40ul of 10% SDS, mix well, then add an equal volume (440u1) of phenol and vortex to mix well.
3) incubate at 65°C for 4mim, cool rapidly in dry ice/ethanol bath until phenol crystals appear, centrifuge at max speed for 2 min,. The supernatant was transferred to a new tube.
4) Add an equal volume of phenol: extract with chloroform, centrifuge for 5 min, transfer the supernatant to a new tube, add 0.1 times the volume of 3mol/L sodium acetate (pH5.3) and times the volume of anhydrous ethanol, and precipitate at 20℃ for 30 min.
5) Centrifuge at 12000 g for lOmin at 4℃, and rinse the precipitate with lml of 80% ethanol. After drying, the RNA was dissolved in 20ul of RNase-free water and stored at -70℃.