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Described here is the protocol for a all information regarding polymerase chain reaction reactions using Q5 DNA polymerase, along with its optimization and troubleshooting. The information is standard reaction protocol and guidelines are largely based on NEB recommendations, and optimization information (additives, troubleshooting protocols) are from elsewhere. –Shyam Bhakta

All information here is also applicable to PCR with Phusion polymerase.

Reaction Setup

Component	25 µL Rxn	50 µL Rxn	Final Concentration
5× Q5 Reaction Buffer	5 µL	10 µL	1×
10 mM dNTPs	0.5 µL	1 µL	200 µM
10 µM Forward Primer or 100 µM	1.25 µL or 0.15 µL	2.5 µL or 0.25 µL	0.5 µM
10 µM Reverse Primer or 100µM	1.25 µL or 0.15 µL	2.5 µL or 0.25 µL	0.5 µM
Template DNA	variable	variable	1 ng–1 pg plasmid/viral. 1 ng–1 µg genomic.
Q5 DNA Polymerase	0.25 µL _{(½ U)}	0.5 µL _{(1 U)}	0.02 U/µL, 1% with 2 U/µL enzyme.
(opt) 5× Q5 High GC Enhancer (opt)	(5 µL)	(10 µL)	(1×)
Nuclease-Free Water	to 25 µL	to 50 µL	-

Assemble all reaction components on ice/cold block in 250 µL "PCR" tubes, unless using Q5 Hot-Start Polymerase which doesn't require keeping cold.
Gently mix the reaction.
Collect all liquid to the bottom of the tube by a quick spin if necessary.
Quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (98°C).

Thermocycling Conditions

	Step	Temperature	Time	Notes
	Initial Denaturation	98°C	30 s–3 min	30 s for most templates (plasmid/linear/E. coli). 1–3 min for complex, or to better lyse cells.
25–35 cycles	Denaturation	98°C	5–10 s	Not >10 s.
	Annealing	*50–72°C	10–30 s	Find T_anl, 3° above T_m of lower T_m primer
	Extension	72°C	10–30 s/kb	10–15 s/kb for simple plasmid/E. coli template. 20–40 s/kb for complex genomic/cDNA template. 30–40 s/kb for >6 kb amplicons.
	Final Extension	72°C	2 min
	Hold	4–10°C	∞

Troubleshooting/optimizing a PCR

Add GC-enhancer or other additives. See Additives section.
Use the touchdown thermocycling protocol. See Annealing section.
Try different annealing temperatures. See Annealing section.

If you need to destroy the non-synthetic template (plasmid), you can add 1 µL DpnI per 50 µL completed PCR, and incubate 37˚C, 15–60 min.

Pausing just after starting the program will pause the protocol after the lid has finished (slowly) heating, after which pausing will pause the program with the block heated to the denaturation temp, step 1.

Reaction

Detailed

Guidelines

Template

Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template are:

DNA	Concentration
DNA Genomic or cDNA	1 ng–1 µg per 50 µL PCR
Plasmid or Viral	1 pg–1 ng per 50 µL PCR

Primers

Oligonucleotide primers are generally 17–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 (in Benchling) can be used to design or analyze primers. The best results typically come from reactions with 0.5 µM each primer.

Buffers

The 5× Q5 Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. For difficult amplicons, such as GC-rich templates or those with secondary structure, the addition of the Q5 High GC Enhancer can improve reaction performance. The 5× Q5 Reaction Buffer is detergent-free and contains 2.0 mM MgCl₂ at at the final (1×) concentration. Shyam deduced that Q5 Reaction Buffer, but not Phusion buffer, contains glycerol, which reduces DNA secondary structure, and tetramethylammonium, which increases primer stringency.

Additives

Amplification of some difficult targets, like GC-rich sequences, may be improved by the addition of 1× Q5 High GC Enhancer. The Q5 High GC Enhancer is not a buffer and should not be used alone. It should be added only to reactions with the Q5 Reaction Buffer when other conditions have failed.

Mg²⁺ concentration of 2.0 mM is optimal for most PCR products generated with Q5 High-Fidelity DNA Polymerase. When used at a final concentration of 1×, the Q5 Reaction Buffer provides the optimal Mg²⁺concentration.

Shyam deduced that Q5 buffer contains glycerol, which reduces DNA secondary structure, and tetramethylammonium, which increases primer stringency.

PCR Additives:

DMSO reduces DNA secondary structure. Use at 3–10% final, adjusting in 2% increments. 10% DMSO lowers T_m 5.5–6°C. DMSO reduces the activity of Taq polymerase.
Glycerol reduces DNA secondary structure. Use at 5–10% final.
Formamide increases the stringency of primer annealing, resulting in less non-specific priming and increased amplification efficiency. Use at 1-10%.
Tetramethylammonium increases the stringency of primer annealing, resulting in less non-specific priming and increased amplification efficiency. Use at 10–100 mM final.
Triton X-100, Tween 20 or NP-40 reduce DNA secondary structure, but can increase non-specific amplification. Use at 0.1–1% final.
Betaine / Betaine·H₂O greatly reduces the high T_m bias of G:C over A:T pairs, reversing the bias slightly at lower T_ms. Especially useful for GC-rich templates. Use at 1–3 M final. Can inhibit amplification of some templates. Don't use betaine·HCl.
1,2-propanediol greatly improves success of GC-rich template amplification. Use at 0.8 M final.
Ethylene glycol greatly improves success of GC-rich template amplification. Use at 1 M final.
7-deaza-2′-deoxyguanosine is a dGTP analogue especially useful for extremely GC-rich templates. Success is reported with up to 83% GC. Use a 1:3 ratio of dGTP:7-deaza-2′-deoxyguanosine.
BSA prevents reaction components adhering to the tube. Use at ≤0.8 mg/mL final.
Tween-20/-40 can neutralize SDS left over from template DNA preparation that would inhibit the reaction. Use at 0.25–1% final.

Sources: Bitesize Bio 1, Bitesize Bio 2, Bitesize Bio 3

Deoxynucleotides

The final concentration of dNTPs is typically 200 μM of each deoxynucleotide. Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and is not recommended for use with uracil-containing primers or templates.

Q5 DNA Polymerase concentration

We generally recommend using Q5 High-Fidelity DNA Polymerase at a final concentration of 20 U/mL (1.0 U/50 μl reaction). However, the optimal concentration of Q5 High-Fidelity DNA Polymerase may vary from 10–40 U/mL (0.5–2 U/50 μl reaction) depending on amplicon length and difficulty. Do not exceed 2 U/50 μl reaction, especially for amplicons longer than 5 kb.

The hot-start formulation, Q5 Hot-Start DNA Polymerase, inhibits the robust exonuclease (and polymerase?) activity of the enzyme, allowing for convenient room temperature reaction setup. The aptamer/inhibitor is released from the enzyme during normal cycling conditions, so no separate activation step is required.

Thermocycling

	Step	Temperature	Time	Notes
	Initial Denaturation	98°C	30 s–3 min	30 s for most templates (plasmid/linear/E. coli). 1–3 min for complex, or to better lyse cells.
25–35 cycles ^_30–35 ^_genomic	Denaturation	98°C	5–10 s
	Annealing	*50–72°C	10–30 s	Find T_anl, 3° above T_m of lower T_m primer
	Extension	72°C	10–40 s/kb	10–15 s/kb for simple plasmid/E. coli template, or <1 kb complex template 20–40 s/kb for complex genomic/cDNA template. 40–50 s/kb for >6 kb amplicons.
	Final Extension	72°C	2 min	^{Holding temp is unnecessary and bad for thermocycler (1).}

If you need to destroy the non-synthetic template (plasmid), you can add 1 µL DpnI per 50 µL completed PCR, and incubate 37˚C, 15–60 min.

Thermocycling Guidelines

Denaturation

An initial denaturation of 30 seconds at 98°C is sufficient for most amplicons from pure DNA templates. Longer denaturation times can be used (up to 3 minutes) for templates that require it.

During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5–10 second denaturation at 98°C is recommended for most templates.

Annealing

Optimal annealing temperatures for Q5 High-Fidelity DNA Polymerase tend to be higher than for other PCR polymerases. The NEB Tm Calculator should be used to determine the annealing temperature when using this enzyme. Typically, use a 10–30 second annealing step at 3°C above the Tm T_m of the lower Tm T_m primer. A temperature gradient across a strip of aliquotted reactions can also be used to optimize the annealing temperature for each primer pair.

2-step PCR

When primers with annealing temperatures ≥ 72°C are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.

Touchdown/Touch-up PCR

See Troubleshooting

Extension

The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid, E. coli, etc.) or complex templates < 1 kb. Extension time can be increased to 40 seconds per kb for cDNA or long, complex templates, if necessary.
When amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50 seconds/kb.

A final extension of 2 minutes at 72°C is recommended.

Cycle number

Generally, 25–35 cycles yield sufficient product. For genomic amplicons, 30-35 cycles are recommended.

PCR product

The PCR products generated using Q5 High-Fidelity DNA Polymerase have blunt ends. If cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is preferred, the DNA should be purified prior to A-addition, as Q5 High-Fidelity DNA Polymerase will degrade any overhangs generated.

Troubleshooting/optimizing a PCR

Recheck primer annealing temperatures and GC-content.
Add GC-enhancer or other additives. See Additives section.
To increase specificity (remove spurious products), use the touchdown thermocycling protocol. See below.
To increase specificity (remove spurious products or obtain a missing product), use the touch-up thermocycling protocol. See below.
To increase specificity (remove spurious products or obtain a missing product), try a range of annealing temperatures. See Annealing section.
Remake your primer stocks.
If strong spurious template, ensure reagents are not contaminated. E. coli genomic contamination is common in cheaply purified polymerase.
If no products at all, ensure polymerase, buffer, and dNTPs are functional in a positive control reaction with known-to-work primers on a trusted template.

Touchdown PCR

To enhance amplification specificity, a touchdown thermocycling protocol can be used, which starts at a higher, stringent T_anl and ramps it down across successive cycles to a steady, permissive T_anl, ensuring high specificity of primer binding in initial products at the elevated T_m, which have a head start in amplification. If the desired band is not visible at all under standard protocol, touch-up PCR must be used. PMID 1861999 PMID 8679209

A typical protocol consists of (after initial denaturation) 10 PCR cycles with the expected T_anl+5°C decrementing 0.5°C per cycle, followed by 25 PCR cycles withT_anl. The T_{anl, i} must be at most the extension temperature. The constant, T_{anl, f} ought to be at least the expected T_anl and can be set as high as the T_anl of the entire primer oligo pair for ensured sustained high stringency. The number of ramping cycles must equal the T_anl elevation divided by the T_anl decrement, and the number of constant T_anl cycles must be ≈25 cycles to ensure sufficient amplification after ramping.

n_{cycles, ramp} = (10–15 cycles) = (T_{anl, i} – T_anl) / ΔT
ΔT = (T_{anl, i} – T_anl) / (10–15 cycles)

Touchdown PCR Thermocycling
	Step	Temperature	Time	Notes
	Initial Denaturation	98°C	30 s–3 min	30 s for most templates 1–3 min for complex, or to better lyse E. coli.
10–15 cycles	Denaturation	98°C	10 s
	Ramping Annealing	T_anl + x	30 s	Ramp by –ΔT every cycle, such that ΔT = x/(10–15 cycles). x = 5–10°C. For x = 7.5°C elevation, ΔT = –0.5°C over 15 cycles.
	Extension	72°C	10–40 s/kb	10–15 s/kb for simple plasmid/E. coli template. 20–40 s/kb for complex genomic/cDNA template. 30–40 s/kb for >6kb amplicons
25 cycles	Denaturation	98°C	10 s
	Annealing	50–72°C = T_anl*	30 s	Find T_anl, 3° above T_m of lower T_m primer
	Extension	72°C	10–40 s/kb	10–15 s/kb for simple plasmid/E. coli template. 20–40 s/kb for complex genomic/cDNA template. 30–40 s/kb for >6kb amplicons
	Final Extension	72°C	2 min	^{Holding temp is unnecessary and bad for thermocycler (1).}

Touch-up PCR

While the touchdown thermocycling enhances amplification specificity by imposing a more stringent initial annealing temperature and ramping to a more permissive T_anl across cycles, touch-up thermocycling does the opposite, by starting at the permissive (expected) T_anl and ramping up across successive cycles to a steady, higher, stringent T_anl, ensuring high specificity of primer binding in later products which are selectively amplified for over successive cycles from non-specific amplicons at the initial permissive T_anl. If the desired band does not appear at the expected T_anl, the initial T_anl can be lower so as to better ensure the correct amplicon is part of the initial selection pool. PMID: 22468135

A typical protocol can consist of (after initial denaturation) 10–15 PCR cycles with the expected (or lower) T_anl,i, incrementing ≥1°C to a final T_anl,f at least 5–10°C higher and as high as that of the entire oligo pair, followed by the remainder of PCR cycles using a T_anl,con equal to T_anl,f. T_anl,f can (perhaps should) be increased 2–5°C for higher threshold stringency, followed by the constant T_anl,con cycles not having that additional increase for better product priming. If the desired T_anl,const is ≥70–72°C, the constant phase can eliminate the annealing step (2-step PCR). If the primers do not have a non-annealing 5′ end or if the entire oligo pair T_anl is not substantially higher than that of just the annealing regions, then the T_anl,i cannot be elevated much without the artificial higher threshold T_anl,f being used for sufficient selectivity. Longer primers can be used or add on non-annealing 5′ ends to select for correct product by providing a T_m advantage to the product.

The 10–15 incrementing T_anl cycles must equal the T_anl elevation divided by the T_anl increment, and the number of constant T_anl cycles should be 20–25 cycles.

n_{cycles, ramping} = (10–15 cycles) = (T_{anl, f} – T_anl,i)/ΔT
ΔT = (T_{anl, f} – T_anl,i)/(10–15 cycles)

Touch-Up PCR Thermocycling
	Step	Temperature	Time	Notes
	Initial Denaturation	98°C	30 s–3 min	30 s for most templates, 1–3 min for complex or to better lyse E. coli.
10–15 cycles	Denaturation	98°C	10 s
	Ramping Annealing	T_anl,i = T_anl(– x) = 45–65°C	30 s	Ramp by +ΔT each cycle, such that T_anl,f is reached in 10–15 cycles i.e. ΔT = (T_anl,f – T_anl,i)/(10–15 cycles) T_anl,f = T_anl,con + y°; y = 0–2–5°C to raise threshold. x ≈ 5° if band is absent in standard PCR Find T_anl,i, 3° above T_m of lower T_m primer
	Extension	72°C	10–40 s/kb	10–15 s/kb for simple plasmid/E. coli template. 20–40 s/kb for complex genomic/cDNA template. 30–40 s/kb for >6kb amplicons
20–25 cycles	Denaturation	98°C	10 s
	Annealing	T_anl,con = 50–72°C	30 s	T_anl,i+5–10°C ≤ T_anl,con ≤ T_{anl,full oligos}
	Extension	72°C	10–40 s/kb	10–15 s/kb for simple plasmid/E. coli template. 20–40 s/kb for complex genomic/cDNA template. 30–40 s/kb for >6kb amplicons
	Final Extension	72°C	2 min	^{Holding temp is unnecessary and bad for thermocycler (1).}

Another kind of touch-up protocol cycles the set of T_anl ramping cycles 4–5 times; it has no constant T_anl phase.

Cyclic Touch-Up PCR Thermocycling
		Step	Temperature	Time	Notes
		Initial Denaturation	98°C	30 s–3 min	30 s for most templates, 1–3 min for complex or to better lyse E. coli.
4–5 cycles	10–15 cycles	Denaturation	98°C	10 s
		Ramping Annealing	T_anl,i = T_anl(– x) = 45–65°C	30 s	Ramp +ΔT°C each cycle, such that T_anl,f is reached in 10–15 cycles i.e. ΔT = (T_{anl, f} – T_anl,i)/(10–15 cycles) T_anl,f = T_anl,con + y° ; y = 0–2–5°C to raise threshold. x ≈ 5° if band is absent in standard PCR Find T_anl,i, 3° above T_m of lower T_m primer
		Extension	72°C	10–40 s/kb	10–15 s/kb for simple plasmid/E. coli template. 20–40 s/kb for complex genomic/cDNA template. 30–40 s/kb for >6kb amplicons
		Final Extension	72°C	2 min	^{Holding temp is unnecessary and bad for thermocycler (1).}

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