Instructions for using DyNAzyme™ DNA Polymerases

1. Standard and long PCR amplifications
2. Long PCR amplifications
3. Notes about reaction components
4. Notes On Cycling Conditions
Quality control of DyNAzyme™ DNA Polymerases
Storage Stability of DyNAzyme™ DNA Polymerases
Specifications of Kit Components
DyNAzyme™ DNA Polymerase Kits (F-550S, F-550L, F-551S and F-551L)

1. Standard and long PCR amplifications

Basic reaction conditions for amplifications up to 10 kb*

• 1x Optimized DyNAzyme™ Buffer (1.5 mM MgCl₂ final conc.)
• 200 µM of each dNTP
• 0.5 µM of each primer (can be optimized between 0.2 - 1.0 µM)
• DNA template
• enzyme 0.02 U/µl
• reaction volume 50 µl

* When amplifying genomic DNA over 3 kb,DyNAzyme™ EXT DNA Polymerase is recommended.

2. Long PCR amplifications

There are several factors affecting the success of long PCR:

• The longer the PCR product is, the more optimization is needed
• Always use hot start
• Use thin-walled reaction tubes
• Use cyclers with fast ramp rates

Basic reaction conditions for PCR amplifications longer than 10 kb

a. 10-20 kb amplifications

• 1x Optimized DyNAzyme™ EXT Buffer / 1x Mg²⁺-free DyNAzyme™ EXT Buffer
• increase the Mg²⁺ concentration to 1.7 mM
• 360 µM of each dNTP
• 0.5 µM of each primer (can be optimized between 0.2 - 1.0 µM)
• DNA template
• enzyme 0.02 U/µl
• reaction volume 50 µl

b. 20 kb and longer amplifications

The conditions are the same as in 2a, with the following changes:
500 µM of each dNTP and 2.3 mM MgCl₂.

Note: Long PCR amplifications are very sensitive to even small variations in the reaction conditions. Therefore the optimal conditions must be determined experimentally.

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3. Notes about reaction components

Mix and centrifuge all tubes before opening to improve recovery. Always pipette DyNAzyme™ DNA Polymerases carefully and gently. The high glycerol content (50 %) in the storage buffer may otherwise lead to pipetting errors.

Enzyme
We recommend 0.5 – 2.0 units of DyNAzyme DNA Polymerases to be used per 50 µl reaction volume. If the enzyme is to be diluted we advise the dilution to be made in 1x reaction buffer or pure H₂O immediately before use. Do not store this dilution.

Mg²⁺ concentration and dNTP concentration
Optimization of Mg²⁺ is critical since DyNAzyme DNA Polymerase is a magnesium dependent enzyme. In addition to DyNAzyme DNA Polymerase, the template DNA, primers and dNTPs bind Mg²⁺. Therefore the optimal Mg²⁺ concentration will depend on the dNTP concentration, the specific template DNA and the sample buffer composition.

If the optimized buffer does not give satisfactory results, optimize the Mg²⁺ concentration between 0.75 and 4.0 mM.

Excessive Mg²⁺ stabilizes the DNA double strand and prevents complete denaturation of DNA, which reduces the yield. Excess Mg²⁺ can also stabilize spurious annealing of primer to incorrect template sites, decreasing specificity. On the other hand, inadequate Mg²⁺ reduces the amount of product. In general, the acceptable Mg²⁺ concentration range narrows as the length of the amplification increases. Usually the optimal Mg²⁺ concentration is 0.5 to 1 mM above the total dNTP concentration for standard PCR and 0.1 - 0.5 mM above the total dNTP concentration for long PCR.

• For standard PCR (0-10 kb), 1.5 mM Mg²⁺ and 200 µM of each dNTP are generally satisfactory.
• For 10 to 20 kb of lambda DNA or ­ 12 kb of genomic DNA; typically 1.7 mM Mg²⁺ with 360 µM of each dNTP.
• For >20 kb of lambda DNA or >12 kb of genomic DNA; typically 2.3 mM Mg²⁺ with 500 µM of each dNTP.

Note: use high quality dNTPs (e.g. F-560)

Note: If the primers and/or template contain chelators such as EDTA or EGTA, the apparent Mg²⁺ optimum may be shifted.

Caution: Repeated freezing and thawing of the buffer can result in precipitation or accumulation of MgCl₂ in insoluble form. For consistent results heat the buffer to 90 °C for 10 min and vortex prior to use if needed. After the first thawing the buffer can also be stored refrigerated to avoid precipitation.

Template
Template preparation becomes particularly important when performing long PCR (>15 kb). The amount of template required depends on the length of the PCR product. For longer amplifications, more template is needed.

General guidelines are: 1 pg - 10 ng/50 µl reaction with low complexity genomes (e.g. lambda DNA); 50-500 ng/50 µl reaction with high complexity genomes (e.g. mammalian DNA).

PCR additives
DyNAzyme DNA Polymerase tolerates the high DMSO concentrations (up to 10 %) needed for opening of difficult templates. Other additives which help DNA denaturation can also be used with DyNAzyme DNA Polymerase (formamide, glycerol, betaine and combinations of these).

We recommend to use co-solvents in the following concentrations:
DMSO 2-10 %, Formamide 2-10 %, Glycerol 5-10 %, or combinations of these.
Recommended starting point is 5 % DMSO.

Note: In high DMSO concentrations the annealing temperature must be lowered, because DMSO decreases the melting point of the primers. For example 10 % DMSO decreases the annealing temperature by 5.5-6.0°C.

Incorporation of nucleotide analogs
DyNAzyme™ I and II DNA Polymerases can utilize dUTP, biotinylated dNTPs, 7-deaza-dGTP, digoxigenin-dUTP, bromo-dUTP, radiolabeled dNTPs and ITP. DyNAzyme EXT DNA Polymerase cannot read dUTP-derivatives or dITP in the template strand and therefore the use of these analogues is not recommended.

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4. Notes On Cycling Conditions

Denaturation
After an initial 1-2 min denaturation at 94°C, keep the denaturation as short as possible (usually 30 seconds or less at 94°C). This is particularly important for long range PCR. Note: the denaturation time and temperature also depend on the ramp rate and temperature control mode of the cycler.

Primer concentration and primer annealing
We suggest the primers to be annealed for one minute or less at the highest temperature that will permit annealing of the primers to the template. A guideline for determination of the annealing temperature is to use a temperature 5 °C lower than the lower Tm calculated by nearest-neighbor method.¹ For long PCR, the primers should be designed to allow a high annealing temperature for maximal reaction specificity (preferably 65°C or higher). If the Tm's of the primers are high enoughFor high complexity or low copy number DNA the primer concentration should be 0.5 - 1.0 µM. For low complexity or high copy number DNA the primer concentration should be 0.3 - 0.5 µM.

Primer extension
The primer extension for standard PCR should be performed at 72 °C, and for long range PCR at 68-70 °C. For shorter PCR amplification (<10 kb) a constant extension time can be used (1 minute per 1.3 - 1.5 kilobases of expected extension product). For long PCR amplification use a constant extension time (start with 1 min per 1.3 - 1.5 kb) only for the first 10 cycles. During the next 15-20 cycles add 20 sec to the elongation time each cycle. To enhance TA-cloning, it may be helpful to include an additional incubation step of 10-30 min at 72 °C at the end.

Cycling instructions for short and long fragments
Cycle program for fragments <10 kb	Temperature	Time	Cycle number
Initial denaturation	92-94 °C	2-4 min	1
Denaturation	94 °C	15 s-1 min
Annealing	45-65 °C *	15 s-1 min	25-35
Extension	72 °C	1 min/1.3-1.5 kb
Final extension	72 °C	5-10 min	1
	4 °C	hold
Cycle program for fragments >10 kb	Temperature	Time	Cycle number
Initial denaturation	92-94 °C	2-4 min	1
Denaturation	94 °C	15 s-1 min
Annealing	55-68 °C *	15 s-1 min	10
Extension	70 °C	1 min/1.3-1.5 kb
Denaturation	94 °C	15 s-1 min
Annealing	55-68 °C *	15 s-1 min	15-20
Extension	70 °C	1 min/1.3-1.5 kb +20 s/cycle
Final extension	70 °C	10 min	1
	4 °C	hold

* Annealing temperature depends on the melting temperature of the primers used. If the Tms of the primers are high enough, annealing and extension can be performed in a single 70/72 °C step.

Quality control of DyNAzyme™ DNA Polymerases

Unit definition
One unit is defined as the amount of enzyme that will incorporate 10 nmoles of dNTPs into acid-insoluble form at 74 °C in 30 minutes under the stated assay conditions.

Unit assay conditions
Incubation buffer:
25 mM TAPS-HCl, pH 9.3 (at 25 °C), 50 mM KCl, 2 mM MgCl₂, 1 mM β-mercaptoethanol, 100 µM dCTP, 200 µM each of dATP, dGTP and dTTP.

Incubation procedure:
20 µg activated calf thymus DNA and 0.5 µCi [-³²P] dCTP are incubated with 0.1 units of DNA polymerase in 50 µl incubation buffer at 74 °C for 10 minutes. The amount of incorporated dNTPs is determined by trichloroacetic acid precipitation.

Exonuclease contamination assay
Incubation of 10 U for 4 hours at 72 °C in 50 µl assay buffer with 1 µg sonicated 3H labeled ssDNA (2 x 10⁵ cpm/µg) released <1 % of radioactivity.

Endonuclease contamination assay
No endonuclease activity is observed after incubation of 10 U of DNA polymerase with 1 µg of DNA or Hind III DNA fragments in assay buffer at 72 °C for 4 hours.

Enzyme stability
Each lot of DNA polymerase is tested for stability under normal storage conditions (-20 °C). The stability of the enzyme is checked at regular intervals for a two-year period.

DNA amplification test
Performance in PCR is tested by the amplification of a 500 bp fragment of DNA and a 6 kb fragment of M13 DNA. DyNAzyme EXT DNA Polymerase is tested by the amplification of 20 kb and 30 kb fragments of DNA.

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Storage Stability of DyNAzyme™ DNA Polymerases

DyNAzyme DNA Polymerases
DyNAzyme DNA Polymerases are stable for at least 1 year during storage at -20 °C. The enzymes themselves are very stable at +4 °C and even at RT, but due to the risk of contamination and possible bacterial growth we do not recommend long term storage in these temperatures. To avoid repeated freezing and thawing, +4 °C is recommended for short term storage up to 1 month.

Storage stability of DyNAzyme EXT DNA Polymerase
DyNAzyme EXT DNA Polymerase is very stable. Long PCR (20 kb) was successfully performed with enzyme samples stored for 2 years at -20 °C, +4 °C and even at room temperature. Enzyme stored at room temperature for 3 years gave a visible band (20 kb) at a concentration of 2 Units/50 µl reaction. Enzyme stored at -20 °C for 3 years gave good bands (20 kb) at different enzyme concentrations (0.5 U, 1 U, 2 U).

¹Breslauer et al., (1986) PNAS 83, 3746-3750)