Download Yeast DNA Prep (Quick) Formosa

8/3/17 DNAPREPD Method 1D page 1
QUICK YEAST DNA PREP
This technique is used for preparing genomic DNA for PCR, for recovering plasmids for transfer to
E. coli, or for Southern Blots. These applications require different variations of the procedure, so the
protocol diverges into three endings.
1. Grow yeast cultures to saturation overnight in 5 ml of YM-1 (+glucose) on a roller at the
appropriate temperature (usually 30°). If recovering a plasmid that can be lost, grow the
strain on a plate to select for retention of the plasmid, then scrape up a large glob of cells for
the inoculation; this works better than using selective liquid medium since the yield of cells
is 5-10 X higher from rich medium.
2. Turn a water bath to 37°C for use in step 11
3. If you are isolating DNA for a Southern blot or otherwise have reason to believe that you
will want to be able to recover the strain for use after you have extracted DNA, spot a wire
loop of the culture (a few µl) to an agar plate (up to 20 can fit on one plate).
4. Spin the cultures for 3 minutes at 2000 rpm in the tabletop centrifuge. Discard the
supernatant and blot any excess liquid from the lip of the tube by touching to a paper towel.
5. Suspend the cell pellet with 1.5 ml of dH2O and pour the suspension into an eppendorf tube.
6. Spin in a microfuge for 5-10 seconds to pellet the cells then discard the supernatant by
either pouring it off or removing with a pipette.
START HERE
1. Add 0.2 ml of quick lysis buffer (add and be sure that pellet is suspended), using the liquid
to try to wash any glass beads away from the lip of the tube. Add 0.15 ml of buffer-saturated
phenol and 0.15 ml of chloroform/IAA, again trying to rinse the glass beads away from the
lip of the tube. Watch out for the chloroform, it will dissolve styrofoam so try not to splash.
If necessary, use a folded kimwipe to wipe the mouth of each tube to make certain the cap
and the lip are free of glass beads or the cap will not form a good seal and the contents will
leak during the next steps. Wear gloves so that you don't get phenol on your skin as this will
cause a serious burn.
2. Add 0.3 ml of glass beads using a cut-off eppendorf tube as a measuring tool. This is a pain
as the beads will get all over the place.
3. Shake the tubes a few times to insure that the cell pellet has been resuspended, then place the
tubes in the vortex machine and vortex for 10 minutes. (Speed 3)
4. Place the tubes in a microfuge and spin for a few seconds to get the liquids away from the
lid, open the tubes, add 0.2 ml TE (or dH2O) to each tube and vortex or shake briefly to mix.
5. Centrifuge in the fixed angle rotor for 5-10 minutes.
6. Transfer 500 µl of the supernatant to a fresh tube containing 1 ml of 95% EtOH with a
P1000 pipettor. Vortex or shake to mix completely.
7. Spin in a microfuge for about 2 minutes. Discard the supernatant.
DNAPREPD Method 1D
For PCR template or transforming into E. coli:
8. Add 1 ml 70% ethanol, vortex, then spin briefly and remove the supernatant.
9. Dry the pellet under vacuum then dissolve the DNA in 50 µl water. Use 1 µl per PCR, 2-3 µl
for transformations.
For Southerns:
14.
Dissolve the pellet with 0.4 ml of TE and 3 µl of 10 mg/ml RNase A. Incubate at 37°C with
occasional vortexing until you can see that the pellet is dissolved (5-15 minutes).
15.
Add 0.2 ml of 30% PEG 8000/1.5 M NaCl solution. Mix well and hold on ice 5 minutes.
16.
Centrifuge in the microfuge (the horizontal rotor is preferred) for 5 minutes.
17.
Discard the supernatant, add 1 ml of 70% EtOH, vortex, then spin for 2 minutes.
18.
Discard the supernatant, and suspend the pellet in 0.4 ml TE. Add 0.2 ml 7.5 M NH4OAc,
mix, then spin 5 minutes in microfuge to remove insoluble material.
19.
Transfer the supernatant to a fresh tube with 0.36 ml isopropanol. Mix well. Spin 5 minutes
and discard the supernatant.
20.
Rinse the pellet with 1 ml of 70% EtOH, spin briefly, discard the supernatant, and dry the
pellet in the Speed-Vac. Dissolve the DNA in 50 µl of TE. Use 3-5 µl/lane for Southern
blots (1-2 µg).
8/3/17 DNAPREPD Method 1D page 3
Quick Lysis buffer:
2% Triton X-100
1% SDS
100 mM NaCl
10 mM Tris·Cl (pH 8)
1 mM EDTA
Stock:
100%
20%
5M
2M
500 mM
Use (for 250 ml)
5 ml
12.5 ml
5 ml
1.25 ml
0.5 ml
Glass Beads:
Sigma G-9268, 425-600 microns
To prepare or recycle glass beads, soak in 1 M HCl for several hours to overnight in a large
beaker. Rinse several times with dH2O (we often use 50-100 mM Tris·Cl, pH 7.5 for the first few
rinses to get the pH into the neutral range more quickly) until the pH is neutral (test with pH paper).
If the amount of glass beads is less than 1 inch deep, cover the beaker with cheesecloth or filter
paper and place in the 37° C room for several days until dry. If more glass beads are being washed,
transfer to a glass tray for the drying step. For faster turnaround, place the beaker or tray in the
heated vacuum oven overnight.
Water saturated phenol
Phenol is purchased as molecular biology grade from the stockroom. Add water to the top of
the bottle, then place at 37° C overnight or until dissolved. Mix well, then allow the phases to
separate. Aliquot the phenol (lower phase) in 50 ml tubes, add a few ml of water, then store at -20°
C. Keep a working stock at 4° C.
Chloroform/Isoamyl alcohol (CHCl3/IAA), 1:25
Add 25 ml of Isopentyl alcohol to 500 ml of chloroform. Mix and store at RT.
RNase A:
Make a 10 mg/ml stock of RNase A (Sigma or stockroom) in TE and place aliquots in a
boiling water bath for 10 minutes. Cool at room temperature, then store at -20°C.
30% PEG/1.5 M NaCl:
For 500 ml, dissolve 150 g polyethylene glycol (MW 8000) and 44 g NaCl in H2O. The
PEG dissolves slowly, and may need to be heated to achieve dissolution.
Why?
The yeast cells are agitated with detergent to dissolve the cell membrane and with glass
beads to enhance the mechanical disruption of the cell wall. The phenol and chloroform induce a
phase separation that denatures proteins and promotes their extraction. These and large cellular
debris are then removed by centrifugation. Nucleic acids and similar materials are precipitated in the
ethanol. The majority of this material is RNA, which is degraded into small pieces by the RNase A.
Precipitation with PEG in high salt removes large molecules but leaves small ones in solution,
separating the DNA from small molecules like the fragments of RNA. The ethanol rinse removes
excess PEG, then ethanol is removed by drying under vacuum.
DNAPREPD Method 1D
This product is clean enough for transforming bacteria, but we have had increased success
with restriction digestions if the DNA is purified further. Most contaminants remaining at this point
(probably proteins) are insoluble in high concentrations of ammonium acetate, but DNA is soluble.
So adding NH4OAC removes impurities, and the supernatant with the DNA can be removed after
centrifugation and the DNA precipitated with isopropanol. Again, rinsing with ethanol removes
excess isopropanol, and the ethanol is removed by treating with vacuum.
8/3/17 DNAPREPD Method 1D page 5
Notes from David Stillman on how to isolate DNA from yeast cells for PCR:
--Resuspend a colony in 30-50 µl of H2O. Use a pipette tip to pick up the cells (not a toothpick).
--Add 1-3 µl of 10 mg/ml zymolyase. Mix well and incubate at 37° 20-30 minutes.
--Boil for a minimum of 5 minutes.
--Spin briefly then transfer to ice.
--Use 1 µl of the supernatant for a 20-50 µl PCR
Suggested PCR conditions:
4' 94°, 1' 55-62°, 5'-10' 72° (1X)
1' 94°, 1' 55-62°, 1' 72° (30X) (extension is 1 min/1000 bp or 10 s per 100 bp)
For E coli:
--Resuspend a colony in 10 µl of LB+antibiotic using a plastic pipette tip (not a toothpick).
--Mix, then transfer 2 µl to a 15 µl PCR (final volume).
--Perform PCR as above.