Standard Operating Protocol for Polymerase Chain Reaction Using HotStar Taq
DNA Polymerase
Prepared by Jennice Lee and Shanthi Wasser
This protocol serves only as a guideline for PCR amplification. Optimal reaction
conditions such as incubation times, temperatures, and amount of template DNA may
vary and must be individually determined.
Please read the complete SOP, especially the safety considerations, before
starting on the experiments. Ensure that all reagents and equipment are available
in sufficient quantities before starting experiment.
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DNA is considered a BSL-1 biological. Standard BSL-1 practices should be used.
Disposable gloves and lab coats are to be used at all times.
All glassware, apparatus and solutions must be sterilized and autoclaved prior to
use
The use of separate set of pipettes and disposable pipette tips with hydrophobic
filters is strongly recommended to reduce cross-contamination
Assume all the nucleic acid samples and modification enzymes as heat labile
and keep on ice unless indicated by protocol
Aliquot the reaction buffers, primer solutions and dNTP mix into smaller volumes
to reduce multiple freeze thaw cycles and contamination
Pulse spin microfuge tubes to ensure that all the added components of reaction
mixtures are mixed at bottom of the tube and to prevent cross contamination
Work bench surfaces must be wiped with 70% ethanol to decontaminate the area
before and after work
Throw all waste into the biohazard bags and when work is finished, waste must
be autoclaved, disposed through the waste management system
In case of contamination, laboratory benches, apparatus, and pipettes can
be decontaminated by cleaning them with a 1/10 dilution of a commercial
bleach solution. Afterwards, the benches and pipettes should be rinsed
with distilled water. Soiled gloves must also be disposed into biohazard
bags.
PCR Protocol Using HotStar Taq DNA Polymerase
1. Set up all reaction mixtures in an area separate from that used for DNA
preparation or PCR product analysis.
2. If required, prepare a dNTP mix containing 10 mM of each dNTP. Store this mix
in aliquots at –20°C.
3. Thaw 10x PCR Buffer, dNTP mix, primer solutions, and Q-Solution.
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It is important to mix the solutions completely before use to avoid localized
concentrations of salts. When using Q-Solution for the first time in a particular
primer–template system, it is also important to perform parallel amplification
reactions with and without Q-Solution.
4. Prepare a master mix according to Table 1
The master mix typically contains all the components needed for PCR except the
template DNA. Prepare a volume of master mix 10% greater than that required
for the total number of PCR assays to be performed. It is extremely important to
include at least one negative control that lacks the template nucleic acid in every
PCR setup to detect possible contamination.
Table 1. Reaction composition using HotStarTaq DNA polymerase and QSolution
Master mix (Total volume 100 µl)
10x PCR Buffer 10 µl
5x Q-Solution 20 µl
dNTP mix (10 mM of each) 2 µl (final conc 200 µM of each dNTP)
Primer A (0.1 µg/µl) 4µl
Primer B (0.1 µg/µl) 4µl
HotStarTaq DNA Polymerase 0.5 µl (final conc 2.5 units/reaction)
Distilled water-Variable volume (Top up to final reaction volume of 100 µl)
Template DNA, added at step 6-Variable volume ~1 µg/reaction
5. Mix the master mix thoroughly and dispense appropriate volumes into
PCR tubes.
Mix gently, e.g., by pipetting the master mix up and down a few times.
6. Add template DNA (about 1 µg/reaction) to the individual tubes containing
the master mix.
7. Program the thermal cycler with the heated lid according to the manufacturer´s
instructions.
8. Each PCR program must start with an initial heat activation step at 95°C for 15
min to activate the HotStar Taq DNA polymerase.
A typical PCR cycling program is outlined below.
For maximum yield and specificity, temperatures and cycling times should be optimized
for each new template target or primer pair.
Initial activation step: 15 min 95°C
(HotStarTaq DNA Polymerase is activated by this heating step)
3-step cycling
Denaturation: 0.5–1 min 94°C
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Annealing: 0.5–1 min 50–68°C (Approximately 5°C below Tm of primers)
Extension: 1 min 72°C. For PCR products longer than 1 kb, use an extension
time of approximately 1 min per kb DNA.
Number of cycles: 25–35
Final extension: 10 min 72°C
Hold 40C
9. Place the PCR tubes in the thermal cycler and start the cycling program.
10. After the completion of the cycling conditions, the tubes can be placed into
the 40C fridge for storage or -200C freezer for long term storage.
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