CHAPTER TWENTY FIVE

Colony PCR
Megan Bergkessel, Christine Guthrie1
Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA
1
Corresponding author: e-mail address: [email protected]

Contents
1. Theory 300
2. Equipment 300
3. Materials 301
3.1 Solutions & buffers 301
4. Protocol 303
4.1 Duration 303
4.2 Preparation 303
5. Step 1 Extraction of DNA from a Colony 304
5.1 Overview 304
5.2 Duration 305
5.3 Tip 305
5.4 Tip 306
5.5 Tip 306
6. Step 2 PCR 307
6.1 Overview 307
6.2 Duration 307
6.3 Tip 307
6.4 Tip 308
6.5 Tip 308
6.6 Tip 308
7. Step 3 Visualization of Product on an Agarose Gel 308
7.1 Overview 308
7.2 Duration 308
7.3 Tip 309
References 309

Abstract
Colony PCR is a method for rapidly screening colonies of yeast or bacteria that have
grown up on selective media following a transformation step, to verify that the desired
genetic construct is present, or to amplify a portion of the construct.

Methods in Enzymology, Volume 529 # 2013 Elsevier Inc. 299

ISSN 0076-6879 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-418687-3.00025-2
300 Megan Bergkessel and Christine Guthrie

1. THEORY
Strategies for genetic manipulation of yeast and bacteria usually
require linking a desired modification with a selectable marker on a con-
struct that is transformed into the organism. The construct can be either a
linear piece of DNA that must then undergo homologous recombination
in order to be stably integrated into the genome, or a circular plasmid that
can be maintained under selection. Following transformation, the organism
is plated onto selective media. A successful transformation will result in the
growth of one to a few hundred colonies on the plate. However, there are
many possible scenarios that can lead to retention of the selectable marker
and subsequent growth of a colony in the absence of the desired genetic con-
struct. Therefore, a screening method is required to distinguish colonies car-
rying the correct construct from colonies carrying the selective marker in
some other context. Colony PCR involves designing PCR primers that will
yield a specific product of known size only if the desired construct is present,
and ideally, a product of a different size if the site of the desired manipulation
remains unaltered. This technique takes advantage of the high sensitivity of
PCR – the small amount of template DNA that is required to give an easily
visualized band on an agarose gel following PCR amplification can be
recovered from a very crude preparation of cells. Colony PCR is thus a
powerful tool for rapidly and easily screening through potentially large num-
bers of colonies to distinguish true positives from false positives. In most
cases it is a superior alternative to the older strategy of growing small cultures
from several colonies, preparing microgram quantities of DNA from each
culture, and performing restriction digests to verify that the desired construct
is present. Additionally, in the case of a cloning strategy that utilizes a step in
one organism to generate a construct that is then recovered, amplified by
PCR, and transformed into a different organism, colony PCR can be used
directly to generate the product for the second transformation.

2. EQUIPMENT
Benchtop microcentrifuge
Vortex mixer
PCR Thermocycler
Microwave oven
Gel electrophoresis equipment
Colony PCR 301

Transilluminator, or other UV light source

Petri dishes, containing colonies to be tested
Micropipettors
Micropipettor tips
0.2-ml thin-walled PCR tubes

3. MATERIALS
Selective culture media, on which colonies are growing
Sodium hydroxide (NaOH)
Purified water (can be distilled and autoclaved, or filtered by Milli-Q or
similar filtration system)
PCR primers
Taq polymerase (or similar thermostable DNA polymerase)
dNTP mix (may be supplied with Taq enzyme, or contains:
dATP
dGTP
dTTP
dCTP)
PCR buffer (may be supplied with Taq enzyme, or contains:
Tris–HCl, pH 8.8
(NH4)2SO4
Tween 20
MgCl2)
Agarose
Tris Base
Boric Acid (H3BO3)
EDTA
Ethidium Bromide
Ficoll 400
Bromophenol blue
Xylene Cyanol
DNA ladder

3.1. Solutions & buffers

Step 1 Sodium Hydroxide (20 mM)
Component Stock Amount/10 ml
NaOH 10 N 20 ml
Add purified water to 10 ml
302 Megan Bergkessel and Christine Guthrie

Step 2 dNTP mix

Component Final concentration Stock Amount/1 ml
dATP 2.5 mM 100 mM 25 ml
dCTP 2.5 mM 100 mM 25 ml
dGTP 2.5 mM 100 mM 25 ml
dTTP 2.5 mM 100 mM 25 ml
Add purified water to 1 ml

10 PCR buffer

Component Final concentration Stock Amount/10 ml
Tris–HCl (pH 8.8) 750 mM 1M 7.5 ml
(NH4)2SO4 200 mM 1M 2.0 ml
Tween 20 0.1% (v/v) 10% 100 ml
MgCl2 15 mM 1M 150 ml
Add purified water to 10 ml

PCR primers
Amounts given for PCR primers assume a working stock concentration of 10 mM.

Step 3 5 TBE buffer

Component Final concentration Stock Amount/liter
Tris base 445 mM N/A 54 g
Boric Acid 445 mM N/A 27.5 g
EDTA 10 mM 0.5 M 20 ml
Add purified water to 1 l

1% Agarose Gel Mix

Component Final concentration Stock Amount/100 ml
Agarose 1% w/v N/A 1g
TBE buffer 1 5 20 ml
Ethidium Bromide 0.5 mg ml 1
10 mg ml 1
5 ml
Add water to 100 ml and heat to boiling in microwave
Colony PCR 303

6 DNA loading dye

Component Final concentration Stock Amount/10 ml
Ficoll 15% w/v N/A 1.5 ml
Bromophenol Blue 0.25% w/v N/A 25 mg
Xylene Cyanol FF 0.25% w/v N/A 25 mg
Add water to 10 ml

4. PROTOCOL
4.1. Duration
Preparation variable
Protocol 3–5 h

4.2. Preparation
PCR primers must be designed and synthesized prior to starting this proto-
col. Commercial primer synthesis has become very economical and widely
available, and the design of appropriate primers is critical to the success of
colony PCR. It is usually worthwhile to design primers specifically for this
purpose rather than use suboptimal primers that may have been designed for
other purposes. Ideally, primers designed to verify the integration of a con-
struct by homologous recombination should include one primer against the
construct and one against the genome adjacent to the desired site of integra-
tion. Colony PCR is most effective for products less than 1 kb in length,
especially when the organism is S. cerevisiae. Larger products have been suc-
cessfully obtained, especially from E. coli colonies, but if the goal is simply to
verify an integration event, smaller products are usually better. It is best to
design primers such that a product is obtained whether the desired construct
is present or not, but the product sizes are unique in each case. Sometimes it
is necessary to design more than one primer set to accomplish this. At a min-
imum, primers that give a product only if the desired construct is present can
be used, but will not distinguish between a failure to obtain the desired con-
struct and a failure of the PCR reaction in the case that no product is
obtained. See Fig. 25.1 for an example.
See Fig. 25.2 for the flowchart of the complete protocol.
304 Megan Bergkessel and Christine Guthrie

Figure 25.1 An example of primer locations for a colony PCR to validate a gene dele-
tion. A construct consisting of a selectable marker flanked by sequences upstream and
downstream of a gene to be deleted is transformed into the organism. Colonies that
grow on selective media presumably express the selectable marker, but some colonies
may have integrated the marker into a random genomic locus (as in colony B) rather
than into the locus of the gene to be deleted (as in colony A). A forward primer
against sequence within the selectable marker combined with a reverse primer against
sequence just downstream of the gene to be deleted (PCR 1) will yield a product only if
the marker has integrated in the appropriate locus. A forward primer against sequence
within the gene to be deleted, combined with a reverse primer against sequence just
downstream of that gene (PCR 2) will yield a product only if the correct integration has
not occurred. Thus, a colony with the correct genotype should yield a band for PCR 1 but
not PCR 2. Each colony should yield a band for either PCR 1 or PCR 2, however – failure of
both reactions would indicate a failure of the PCR protocol, rather than a negative result
for the presence of the desired construct. All the three PCR primers should have a similar
melting temperature, preferably above 60  C, and should not be complementary to
each other. Product sizes should be 200 bp–1 kb.

5. STEP 1 EXTRACTION OF DNA FROM A COLONY

5.1. Overview
Although PCR requires only a very small amount of template DNA, it is
more successful, especially for S. cerevisiae, if cells are treated to break down
the cell wall and allow the DNA to escape.
Colony PCR 305

Figure 25.2 Flowchart of the complete protocol, including preparation.

5.2. Duration
15 min
1.1 Start with large (2 mm) colonies. Colonies can be restreaked into a
larger patch on a second plate of selective media if they are very
slow-growing, or if the first plate is crowded with colonies.
1.2 For each colony to be screened, pipette 20 ml 20 mM sodium hydrox-
ide into a 0.2-ml thin-walled PCR tube. Using a pipettor tip, scrape up
about half of a colony and pipette up and down in the 20 ml sodium
hydroxide to disperse the colony in the liquid. Label the colony on
the plate so that positive colonies can later be identified.
1.3 Incubate tubes for 8 min at 100  C in a PCR machine, with a heated lid.
1.4 Briefly spin the tubes in a microfuge to collect liquid at the bottoms of
the tubes. Mix each tube vigorously using a vortex mixer at top speed
for about 15 s.
1.5 Spin tubes in a microfuge for about 30 s at top speed to pellet the
cell debris.
1.6 Use the supernatant as the template DNA in a PCR reaction, as
described in Step 2.

5.3. Tip
Because PCR is so sensitive in detecting very small amounts of DNA, it is possible for
DNA from dead cells that have failed to grow on the selective media to be amplified in
306 Megan Bergkessel and Christine Guthrie

colony PCR. Depending on how the primers for the colony PCR have been designed,
this can lead to a false band in a negative control reaction. Usually these false bands are
faint. This can be minimized by restreaking colonies onto a fresh plate of selective
media and using these cells for colony PCR, but adding this step costs 1–2 days.

5.4. Tip
This step is probably not necessary if the organism is E. coli. Good results have been
observed when a small amount of an E. coli colony is scraped up with a pipetor tip and
dispersed directly into a PCR reaction mix. This also has been effective with some
other yeast species and may even sometimes be effective for S. cerevisiae. This step
will not decrease the efficacy of the PCR, however, and probably helps the DNA
escape the cell wall.

5.5. Tip
A single E.coli colony can be used for simultaneous PCR amplification and overnight
growth. First, disperse some of the scraped colony into the PCR master mix. Disperse
the remainder into a tube containing 1–2 ml of LB medium plus the appropriate anti-
biotic and grow overnight at 37  C with shaking.
See Fig. 25.3 for the flowchart of Step 1.

Figure 25.3 Flowchart for Step 1.

Colony PCR 307

6. STEP 2 PCR
6.1. Overview
A standard PCR reaction is used to amplify the product of interest from the
DNA that has been extracted from the colony in Step 1. A typical set of PCR
conditions is described, but any commercially available thermostable poly-
merase and its supplied buffer should work (see General PCR).

6.2. Duration
2–2.5 h
2.1 Make a PCR master mix, and keep on ice:
per sample:
16.25 ml purified water
2.5 ml 10 PCR buffer
2.5 ml dNTP mix
1.25 ml forward primer
1.25 ml reverse primer
0.25 ml 100 Taq
2.2 Pipette 24 ml of the PCR master mix into 0.2-ml thin-walled PCR
tubes. Keep tubes on ice.
2.3 Pipette 0.5–1 ml of the supernatant from each sample into a PCR tube
containing PCR master mix.
2.4 Use a thermocycler to carry out PCR. Typical reaction conditions are
as follows, for a product up to 1 kb in length:
5 min at 95  C
repeat 30 times:
15 s at 95  C
15 s at 57  C
1 min at 72  C
finish with:
5 min at 72  C

6.3. Tip
If skipping Step 1, increase the amount of water per reaction by 1 ml, and pipette
25 ml PCR master mix into a 0.2-ml thin-walled PCR tube for each colony to
be tested. Then disperse the colonies directly into this liquid. Be sure to label the col-
onies on the plate so positives can later be identified.
308 Megan Bergkessel and Christine Guthrie

Figure 25.4 Flowchart for Step 2.

6.4. Tip
If using a HotStart Taq polymerase, it is not necessary to place the tubes on ice while
setting up the reactions.

6.5. Tip
When preparing a PCR master mix, make enough for the number of reactions plus
0.5–1 extra.

6.6. Tip
If the goal of the colony PCR is to produce a sufficient quantity of a construct for a sub-
sequent transformation step into a different organism, a larger volume PCR can be carried
out. It may also be advisable to use a higher fidelity thermostable polymerase, or mixture
of polymerases, since any mutations acquired during this PCR step could be deleterious in
future cloning steps. Check the supplier’s manual if you use a high-fidelity thermostable
polymerase. The extension is often carried out at a lower temperature.
See Fig. 25.4 for the flowchart of Step 2.

7. STEP 3 VISUALIZATION OF PRODUCT ON AN

AGAROSE GEL
7.1. Overview
The PCR products that have been generated are now separated by size on an
agarose gel containing a DNA-intercalating dye such as ethidium bromide
(see Agarose Gel Electrophoresis).

7.2. Duration
1.5 h
3.1 Cast the agarose gel mix into a gel tray after boiling in a microwave
oven to completely dissolve the agarose. Take care while heating the
Colony PCR 309

Figure 25.5 Flowchart for Step 3.

gel mix not to allow it to boil over. This can be done while the PCR is
running. Fill the gel tank with 1 TBE, the buffer in which the gel will
be run. Note that ethidium bromide is toxic. Wear gloves when han-
dling the gel and take care to dispose of it properly.
3.2 Add 2 ml DNA loading dye to 10 ml of each sample. Load onto the gel.
Load DNA ladder (as commercially supplied, or about 500 ng DNA in
a volume of about 10 ml, containing loading dye) in one lane.
3.3 Run the gel at 100 V for 45 min, or until xylene cyanolhas run about
three quarters the length of the gel.
3.4 Use a UV light box to visualize the DNA bands on the gel.

7.3. Tip
If the goal of the colony PCR is to produce a sufficient quantity of a construct for a
future transformation step, run only a small fraction of the PCR product on a gel to
verify that the product is the correct size. If a single band is obtained in high enough
amounts, the PCR mixture can be used directly for a transformation of some organ-
isms, including S. cerevisiae.
See Fig. 25.5 for the flowchart of Step 3.

REFERENCES
Referenced Protocols in Methods Navigator
Agarose Gel Electrophoresis
General PCR