Preparation of yeast total RNA

Summary

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
High speed centrifuge

Move

I. Materials and equipment

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℃.

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.

https://www.aladdinsci.com/