A) Phenol saturated with 10 mM Tris, 100 mM NaCl, 1mM EDTA, pH ~7.5
This solution was pHed by addition of 6M NaOH while stirring and measuring the pH of the buffer with
a pH meter.
B) Chloropane 49% Phenol
2% Iso-amyl Alchohol
C) 50 mM Tris pH 8, 10 mM EDTA
1) Grow 500 mls of Yeast in YPD to OD 1-2. ( 40 ul into 500 ml reaches OD600 ~1 in 20 hrs.)
Add extra adeneine (1x) if your strain is an ade2 mutant or your RNA will be red.
*We have also used 1 liter of a stationary phase cultures in this protocol, you get 10 mg of RNA/ liter of cells.
This is actually easier because you can store the pelleted cells at -70° and make RNA at your liesure.
2) Pellet cells and resuspend in 22 mls of 50 mM Tris, !0 mM EDTA pH 8, place in a 250 ml capped flask.
Cell pellets can be frozen in liquid N2 and stored before resuspension if desired.
3) Add 2 mls of 10% SDS and 25 mls of phenol preheated to 65oC, and shake at 65oC for 6 minutes. It is best
to shake mechanically in a waterbath at 65oC.
4) Chill in a dry ice ethanol bath or liquid N2 by swirling until phenol crystals form on the side of the flask.
5) Spin in 30 ml Sarstat tubes for 5 minutes at 5000 rpm in a Sorvall centrifuge.
6) Remove supernatant and add to another 250 ml flask containing 25mls of preheated phenol
and shake at 65oC for 6 minutes. Chill in dry ice ethanol bath again.
7) Spin at 5000 rpm for 5 min. again to separate the phases. Remove the supernatant with RNAse free
pasture pipets and add to 25 mls of chloropane. Shake at room temperature for three minutes and spin
in a table-top centrifuge for 5 minutes at maximum velocity.
8) Remove the supernatant and EtOH precipitate by the addition of 2 volumes of EtOH.
This entire protocol can be scaled down to prepare RNA from 10 mls of cells
using 50 ml flasks and eppindorf tubes.
Remember: 1 OD260 = 40ug/ml for RNA
1. Pick a medium sized colony (about 3mm in diameter) into a200ul of
300 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA, 0.1% SDS in a 1.5 ml Eppendorf tube. Vortex by hand.
2. Add glass beads (0.45 nM diameter) until just below the level of the liquid.
3. Mix vigorously on a vortex mixer for 1 minute. Phenol extract the sample containing the glass beads with an equal volume of PCI (Phenol : Chloroform : Isoamyl, 25:24:1). Repeat the extraction until no interface is observable, then extract with one volume of Chloroform.
4. Ethanol precipitate by addition of 2 volumes EtOH. Wash the pellet with 1ml of 80% ethanol.
5. Resuspend pellet in 100 ul of TE.
6. Transform E. coli with varying amounts of the DNA. 1 ul should give numerous colonies depending upon the competence of the bacteria.
1. Prepare 10X MOPS buffer:
4.10g anhydrous NaAc
3.72g disodium EDTA
Q.S. to 1 liter with DEPC H2O
Adjust the pH to 6-7 by adding
from 10 to 12 NaOH pellets
Store at RT under wraps
2. Prepare gel:
Combine 2g Agarose (˜1.2%)
113ml DEPC H2O
Boil in microwave and cool to 60°C
Add 16.6ml 10X MOPS buffer
and 25ml Formaldehyde
Mix, pour and let stand 30′
3. Prepare 1X MOPS running buffer and prerun the gel at low voltage (5V/cm) for an hour
4. Resuspend RNA in as little DEPC H2O as possible (but at least 15µl) then combine 5µl of the RNA suspension with the following in order:
10µl of deionized Formamide
2µl 10X MOPS buffer
(Running dye is prepared by mixing approximately 1µl of Xylene cyanol FF and Bromophenol Blue powders in 2µl of water to make a colloid. Add the dye to the samples by picking up a very small amount on a Pipetman™ tip and mixing this in the samples.)
5. Heat the sample to 65°C for 15′ and then cool the mixture on ice. A very small amount of a 10 mg/ml EtBr solution is added from the end of a Pipetman™ tip.
6. Load the sample and run at 100V (24mA) or 30V for ON runs. (DO NOT SUBMERGE THE GEL UNDER MORE THAN 1-2 mm OF BUFFER)
1. Grow 1 liter of yeast cells in YPD to stationary phase.
2. Pellet and wash 2x and resuspend in zymolyase buffer.
3. Zymolyase treat for 1 hour, check for speroblasting
4. Gently pellet spheroblasts and resuspend in 12.5 mls of lysis buffer
50 mM Tris pH 8.0
50 mM EDTA pH 8.0
1 % Sarkosyl
5. Add 800 ul of 10 mg/ml RNAse A solution, 1 h at 37°C
6. Add 1 ml of 40/mg/ml protienase K 1 h at 37°C
7. CHCl3 extract
8. Add CsCl to 1.71 g/ml and spin.
1) Yeast cells are grown to OD 0.4 in YPD pH 3.9 (pHed with HCl) and a-factor is added to a final concentration of 2.0 ug/ml.
2) Cells are allowed to incubate for 1 hour at 30°C at which time another 1ug/ml a-factor is added and cells are incubated for a further hour. Cells should be monitored for bud percentage periodically
3) At 2 hours >90% of the cells should be unbudded. Cells should be pelleted, washed 2x with YPD at normal pH, and resuspended in YPD and time points taken every 10 minutes.
1. All strains do not behave identically under these condition, so pilot experiments should be performed monitoring budding.
2. Cells rush through the first cell cycle after a-factor synchronization.
3. For time points, we generally spin down the cells quickly and freeze the pellets in liquid nitrogen so that RNA can be prepared at the same time.
4. This is for a wild type strain. If you are Dsst2, you will require less a-factor. The pH inactivates the BAR1/SST1 protease and resuspending back into YPD reactivates it and helps with synchronizing re-entry into the cycle.
5. We originally did this with 1 liter of cells and took 50 ml time points. This gave us more RNA than we needed. It can be scaled down to 200 mls and probably work fine.