RNA extraction

 

1.      Grow yeast cells to the desired growth rate.

2.      Harvest cells by centrifugation at 4000 rpm for 3 min.

3.      Resuspend in a very small volume of growth medium.

4.      Pipette in the suspension and release as individual drops directly into liquid nitrogen.

5.      Frozen cell drops can be stored at –80°C.

6.      Pre-cool a 5ml teflon vessel in liquid nitrogen.

7.      Add a 7 mm tungsten carbide bead and frozen cells equivalent to about 15 OD600 units of cells.

8.      Close flask with pre-cooled cap and shake it using a micro-dismembrator (Braun, Melsungen) at a frequency of 2600 rpm for 2 min.

9.      Transfer the resulting frozen powder into Trizol ReagentTM (1ml of reagent/OD600 unit).

10.  Homogenise by vortexing for 1 min, and keep at room temperature for 5 min.

11.  Transfer the mixture to a Correx tube and centrifuge at 12000xg for 10 min at 4°C.

12.  Transfer clear supernatant to a fresh falcon tube, add 2/10 volume of chloroform, shake vigorously for 15 s and keep at room temperature for 3-10 min.

13.  Centrifuge at 12000xg for 5 min.

14.  Transfer aqueous layer to a fresh correx tube and precipate RNA with 0.5 ml of isopropanol per ml of reagent, vortex, and keep the samples at room temperature for 5-15 min.

15.  Centrifuge for 10 min at 4°C at 12000xg.

16.  Remove isopropanol carefully and wash pellet twice with 1ml of 70% EtOH and centrifuge as above.

17.  Air-dry pellet for a couple of minutes.

18.  Resuspend RNA pellet in 500 ml H20 (DEPC treated), vortex and keep at 65°C for 5 min.

19.  Transfer into an eppendorf containing 1 volume of LiCL buffer and precipitate at –20°C for at least 1h.

20.  Centrifuge at max speed for 30 min at 4°C.

21.  Wash pellet twic with 70% EtOH.

22.  Dry pellet and dissolve in a small volume of DEPC treated H20.

 

 

Buffer and solutions :

      Trizol Reagent (GibcoBrL – cat no : 15596-018)

      Chloroform

      Isopropanol

      70% ethanol

      DEPC treated H20

      LiCl-buffer       4M LiCl

                             20 mM Tris/HCl pH 7.5

                             10 mM EDTA

 

 

 

First strand cDNA synthesis :

 

1.      Mix 500 mg of oligo dT15 with 12.5 ml of total RNA (equivalent to 20-30 mg, but the volume depends on the amount of radio-label used), and incubate at 70°C for 10 min.

2.      Cool briefly on ice and place at 42°C.

3.      Add and mix :        6 ml 5x first strand buffer

3 ml 0.1 M DTT

1.5 ml AGT+C mix

5 ml 33 P –dCTP (50 mCi) (this amount can be doubled)

1 ml Superscript RT (200 U)

4.      Incubate at 43°C for 1-2h.

 

Alkaline hydrolysis of RNA :

 

5.      Add           1 ml 1% SDS

1 ml 0.5 M EDTA pH 8.0

3 ml 3M NaOH

6.      Incubate at 65°C for 30 min.

7.      Keep at room temperature for 15 min.

8.      Add           10 ml 1M Tris/HCl pH 8.0

3 ml 2N HCl

 

Isopropanol precipitation :

 

9. Add             5 ml NaOAc pH 5.3

                        5 ml tRNA 10 mg/ml

                        60 ml isopropanol

10    Precipitate at –20°C for 30 min.

11    Centrifuge at max speed for 30 min.

12    Redissove cDNA in 100 ml H20

13    Or alternatively use a QLAquick Nucleotide removal Kit.

 

Buffer and solutions :

      DEPC-treated Ho

      Oligo dT15 (500 mg/ml)

      5x first strand buffer (Gibco BRL) :      250 mM Tris/HCl pH 8.3

                                                                 375 mM KCl

                                                                 15 mM MgCl2

 

0.1   M DTT (Gibco BRL)

AGT+C mix :   16 mM dATP

                        16 mM dGTP

                        16 mM dTTP

                        100 mM dCTP

 

33P-dCTP

SuperScript RT (200U/ml) (Gibco BRL)

1% SDS

0.5 M EDTA ph 8.0

3M NaOH

1M Tris/HCl pH 8.0

2N HCl