Yeast Colony
PCR
adapted from
www.fhcrc.org/labs/hahn/methods/mol_bio_meth/pcr_yeast_colony.html
Chris Tachibana May 2004, updated
July 2005
¥ No more than 1 microliter of the
crude DNA should be added to the 50 microliter PCR reaction because the SDS in
the DNA prep can inhibit the PCR reaction.
¥ Works best if the product is
less than 1.0 kb.
¥ Use Platinum Taq, or other hot
start Taq. (As of 2005, we are
mostly using Phusion Polymerase)
¥ Start by choosing a DNA prep
method: Method 1 makes a small genomic DNA prep that can be used for several
reactions and stored; Method 2 lyses the cells right in the PCR reaction.
------------------------------------------------------------------------
Method 1:
1. Prepare Yeast DNA (this
works best with fresh yeast plates)
¥ Use a pipetman tip to transfer
yeast cells (about this much: ¥ ) to 30
microliters of 0.2% SDS. JuanJo
had success using a pellet of 3 x 106 cells from a liquid
culture.
¥ Vortex ~15 seconds
¥ Heat in hot block for 4 min at
90 deg.
¥ Spin in microfuge 1 min. Remove
supernatant to a new tube. Store at -20 degrees.
2. PCR Mix
PCR can be done in 50µl or 20µl
volumes. Either way, use as little
DNA as possible (0.5µl). It works
best with the Phusion polymerase but you can also do it with Platinum Taq
polymerase.
a) Phusion 50µl per tube
¥ 10 µl 5X buffer HF
¥ 34 µl water (for 2 primers)
or
32 µl water (for 3 primers)
¥ 2 µl each primer from a 5µM
stock
¥ 1 µl 10mM dNTPs
¥ 0.5 µl Phusion Enzyme
49.5 µl per tube + 0.5µl DNA, use
the "Col-Phu" program in the MJResearch PCR machines
b) Phusion 20µl per tube
¥ 4 µl 5X buffer HF
¥ 13.3 µl water (for 2 primers)
or
12.5 µl water (for 3 primers)
¥ 0.8 µl each primer from a 5µM
stock
¥ 0.4 µl 10mM dNTPs
¥ 0.2 µl Phusion Enzyme
19.5 µl per tube + 0.5µl DNA (or
less), use the "Col-Phu" program in the MJResearch PCR machines
c) Platinum Taq (50µl
reactions)
5 microliters 10x colony PCR
buffer
1.5 microliters 50 mM MgCl2
1 microliter 10 mM mix of dNTPs
10 pmoles of each primer (~100 ng
of a 25 mer oligo, 2 microliters of a 5 µM stock)
2 microliters 25% Triton X-100
0.3 microliter Platinum Taq
Polymerase [Invitrogen] (5 u/microliter)
H2O to a final volume of 49
microliters (35.2 microliters)
Aliquot 49 µl into PCR tubes.
Add mix to PCR tubes on ice
containing 1 microliter of the crude DNA prep from above.
Go to "PCR cycle
profile" below.
Method 2:
1. PCR Mix
Combine the following
components on ice (per 50 µl reaction):
5 microliters 10x colony PCR
buffer
1.5 microliters 50 mM MgCl2
1 microliter 10 mM mix of dNTPs
10 pmoles of each primer (~100 ng
of a 25 mer oligo, 2 microliters of a 5 µM stock)
2 microliters 25% Triton X-100
H2O to a final volume of 49
microliters (35.5 microliters)
Aliquot 49 µl into PCR tubes.
Touch a yeast colony with a
sterile 20µl pipet tip and put the cells directly into a PCR tube with PCR
mix. The mix should be slightly
cloudy but not dense with cells.
Heat 8 minutes, 94 degrees
(program 97 in the Robocycler).
While they are heating, dilute the Taq polymerase.
Add 1µl of Platinum Taq Polymerase
diluted 1:2 in 1X colony PCR buffer.
You can do this without removing the tubes from the Robocycler.
Go to "PCR cycle
profile" below.
3. PCR profile
a) for Phusion Taq
98 deg 30 sec
98 deg 5 sec
52 deg 10 sec
72 deg 15 sec
repeat steps 2-4 for 34 additional
cycles
72 deg 5 min
4 deg hold
b)for Platinum Taq
95 deg 1 min
95 deg 30 sec
52 deg 1 min (optimum annealing
temp varies according to primiers)
72 deg 2 min
repeat steps 2 through 4 for a
total of 35 cycles
72 deg 6 min
hold at 4 deg
Load 20µl microliters to agarose
gel for assay.
For DNA sequencing analysis of
product, purify using QIAquick PCR purification kit (Qiagen), eluting the
product in 30 microliters. Use ~6 microliters for DNA sequencing analysis.
------------------------------------------------------------------------
MATERIALS
0.2% SDS
10X Colony PCR buffer (if using
Platinum Taq)
0.13 M Tris-HCl pH 8.5 87µl
of 1.5 M
0.56 M KCl 187
µl of 3 M
726
µl of water
50 mM MgCl2
10 mM dNTP mix
25% Triton X-100 (if using
Platinum Taq)
PCR primers ~25 bases in length
specific for the region of interest with annealing temp 55-60 deg.