PCR Amplification
[This protocol is a general guide to PCR design and set-up; each polymerase will have a slightly
different set-up of ingredients, temperature preferences, extension times, etc.]
PCR using “KOD Hot Start Polymerase”
1. Standard PCR (no tails). For oligos that do not have over-hanging “tails,” or for a standard
diagnostic PCR (to visualize only, and not used further), the following protocol should be used
for KOD.
2. Set up PCR components as follows. Can set up at room temp. Shown is for 50 uL reaction:
(i) 5 uL of 10x Buffer*
(ii) 5 uL of dNTP mix
(iii) 3 uL of Mg2+**
(iv) 5 uL of Forward primer (working dilution)
(v) 5 uL of Reverse primer (working dilution)
(vi) 0.5-1.0 uL of KOD enzyme***
(vii) DNA template****
(viii) water to final volume
*Thaw supplied components and primers and vortex to mix. Buffers can sometimes have
precipitates, vortex to mix well. Store at -20°C. (Primers may be stored at 4°C fridge, but longterm storage should be in freezer at -20°C).
**Some PCR kits already have Mg2+ added in the buffer. KOD has it separate.
***The amount of enzyme should vary depending on needs. For standard PCR, 1 uL is
recommended per 50 uL reaction. For diagnostic, lower the overall volume of the reaction to
save costs (15-25 uL reaction will require less components and less KOD). For IVLs, or for
integration PCRs where 50 uL is needed, then use 0.9-1.0 uL KOD/50 uL reaction. Keep frozen
until needed and use cold block to keep enzyme cold (replace to freezer once done!).
[Note: A “Master Mix” is often very useful depending on the needs of the experiment. In general,
a Master Mix includes water + dNTPs + buffer + Mg2+. For experiments that all use the SAME
primers, the F and R primers can also be included in the Master Mix (this saves a lot of time and
pipette tips!). In the case of a master mix, combine all components (NEVER KOD and NEVER
DNA!) into a tube, vortex to mix, then aliquot the total combined volume to each PCR tube.
****DNA template amount varies depending on the source. For Genomic Preps (chromosomal
DNA) from yeast, use 2.0-2.3 uL of DNA per 50 uL reaction. For DNA Plasmid Minipreps, use
0.1-1.0 uL. For use in in vivo ligation (IVL) protocols, and if the template DNA is from a yeast
plasmid (e.g. pRS315 series or other CEN-based vector), then a 1:10 dilution of the plasmid
miniprep can be made in water, first, and then 0.5 uL can be added to the PCR reaction).
3. The final steps of setting up the PCR should be addition of the template DNA, and then
addition of the KOD polymerase to each tube. [Note: the KOD, or any polymerase, will be in a
glycerol containing solution and will be viscous; pipette with great care to transfer the exact
amount with the appropriate pipette size/type.]
4. To ensure all the volume is at the bottom of the PCR tube, centrifuge very briefly at low speed
(5,000 rpms for 5 seconds) and carefully handle tubes.
5. Set up PCR program (example shown; this varies depending on the experiment):
1. 95°C for 2 minutes
2. 95°C for 15 seconds
3. 53.5°C* for 30 seconds
4. 72°C for 40 seconds**
5. Go to Step 2, 29x***
6. 72°C for 10 minutes
7. Hold at 10°C forever
*The Annealing temperature can be altered depending on the F and R primer. In general, a very
rough estimate of annealing temp can be the average of the Tm of the F and R primers minus 5.
In general, run at between 51-54°C for annealing.
**The extension time is 30-40 seconds per 1,000 bps for standard PCRs for plasmid template.
For genomic template, use 1 minute per 1,000 bps. For small fragments, use 30 seconds total
extension time.
***25-30 total cycles is sufficient for diagnostic PCRs.
6. PCRs can be stored at 4°C (fridge) until needed. Long-term storage is not recommended.
7. Run a small portion (or the entire reaction if diagnostic) by DNA gel electrophoresis to
visualize band.
8. PCR with Tails (2-step): For PCRs where one or both oligos contain extended “tails” of any
length which are NOT present on the template DNA, a modified PCR reaction set-up should be
used. Because the initial primers will anneal via their “base” sequence matching the template
(and thus, the overhanging tails match NOTHING initially), a 2-step reaction should be used.
[Note: it is recommended that in primer design, the “base” portion (matching the template) of
each primer should shoot for a Tm of roughly 60°C still regardless of the length.]
9. The PCR reaction should be set up exactly the same as a “standard” reaction.
10. The program for a 2-step PCR should be as follows:
1. 95°C for 2 minutes
2. 95°C for 15 seconds
3. 53.5°C* for 30 seconds
4. 72°C for 40 seconds
5. Go to Step 2, 5x
6. 95°C for 15 seconds
7. 58.5°C** for 30 seconds
8. 72°C for 40 seconds
9. Go to Step 6, 24x
10. 72°C for 10 minutes
11. Hold at 10°C forever
*The initial annealing temperature should be the “standard” one used to pair with the “base”
portion of the oligos (assuming Tm of 60°C). This is repeated for 5 or 6 initial cycles to allow for
“priming”.
**The second step is the same with the annealing temperature increased by 5°C (regardless of
the actual Tms of the primers + tails). I always go with 53.5°C and 58.5°C for 2-steps. These
temperatures may be varied slightly given different templates/needs.
11. Quikchange PCR mutagenesis: This is a special case where a mutation (or two or three)
is needed and, the entire template is PCR amplified. Please see the protocol on Quikchange
mutagenesis PCR.
12. Inverse PCR: This is a second, similar special case PCR reaction where several bases or a
stretch of bases can be incorporated into position by PCR amplifying the entire vector. Pros, can
introduce several codons, or tags at once. Cons, requires standard ligation protocol, requires T4
PNK treatment of the primers (to add phosphates), not as reliable as IVL in yeast. See protocol
on Inverse PCR.
[Notes: Failed PCRs: The major sources of difficulty for PCR are (i) choice of template and (ii)
primer design. Today, most polymerases and thermocyclers are so advanced that the issue is
rarely the temperature, buffers, or conditions. The DNA template and/or the primer design
account for the majority of errors or difficulties. Trouble-shooting the PCR reaction should not
(initially) involve other temperatures or times on the machine or conditions (Mg2+, DMSO),
rather, a closer look at the primers and template should be done first.]
[Notes: Genomic DNA: PCRs of genomic DNA are more “difficult” than from purified plasmid
DNA. It is not recommended that PCRs contain long extended “tails” (2-step) using genomic
DNA as a template. Instead, a standard PCR can be used to clone the fragment/gene into a
vector (e.g. TOPO II), and then subsequent PCRs can modify or prime fragments from this
vector (which can also be preserved in the collection). Additionally, some loci and some genes
are just inherently difficult to PCR. The position of the locus matters greatly (as compared to the
sequence of the primer). For instance, for the CDC10 gene, a F primer at +800 upstream of the
START is difficult to prime with; rather, a +698 F primer is used. Similarly, positions upstream of
406 5’ UTR for the CDC11 gene are difficult to PCR amplify, this +300 and +400 primers are
used to interrogate that locus. If an initial (and new) locus is being amplified, it is recommended
that several primers around the promoter/terminator be tested. Some loci are just notoriously
hard to amplify.]
[Notes: Plasmid DNA for use in IVL or integration: If the template DNA happens to be (or has
to be) from a yeast CEN-based plasmid (e.g. pRS315 or another IVL vector from the collection),
several modifications to the protocol should be done to minimize background: (i) dilute the initial
plasmid miniprep DNA 1:10 or 1:20 in water prior to adding a small amount to the reaction, (ii)
Dpn1-treatment (straight into the PCR tube) overnight to digest the plasmid DNA template, (iii)
screening subsequent colonies for sensitivity to the original template DNA plasmid’s selection
marker (N/A if this is the same as the newly designed vector).]