Preparation of yeast total RNA
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℃.