Taq DNA Polymerase 2x Master Mix

1.5 mM MgCl2 final concentration

Protocol
This protocol serves as a guideline for primer extensions.
Optimal reaction conditions such as incubation times,
Cat. No.: A140301 temperatures, and amount of template DNA may vary and must
be determined individually.

Size Taq DNA Polymerase 2x Master Mix, Notes:

Cat. No.
Reactions 1.5 mM MgCl2  Set up reaction mixtures in an area separate from that used
ID No. 5200100 for DNA preparation or product analysis. Work on ice at all
-
Cap colour Red times.
A140301 100 2 x 1.25 ml
 The final MgCl2 concentration of this 2x Taq Master Mix is
1.5 mM. In some applications, more than 1.5 mM MgCl2 is
required for best results. Use 25 mM MgCl2 (see Additional
2+
Products) to adjust the Mg concentration according to
table 1.

Table 1. Additional volume (µl) of MgCl2 per 50 µl reaction:

Final MgCl2 conc.
1.5 2.0 2.5 3.0 3.5 4.0 4.5
Key Features in reaction (mM)

 High performance thermostable DNA polymerase Volume of 25 mM MgCl2 0 1 2 3 4 5 6

 Significantly reduced set up time
1. Thaw Taq 2x Master Mix and primers. It is important to
 Increased reproducibility
thaw the solutions completely and mix thoroughly before
 Diminished risk of contamination
use to avoid localized concentrations of salts. Keep all
 No proofreading – lacks a 3'5' exonuclease activity components on ice.
 Ideal for TA cloning – leaves an A overhang
2. Set up each reaction. Table 2 shows the reaction set up for a
Taq DNA Polymerase 2x Master Mix is a ready-to-use 2x reaction final volume of 50 L. If desired, the reaction size may be
+
mix with the Ampliqon Taq DNA polymerase, the NH4 buffer scaled down. Use 10 µl of the Taq 2x Master Mix in a final
system, dNTPs and magnesium chloride present. Each reaction volume of 20 µl.
requires 25 µl of the 2x Master Mix. Simply add primers,
template and water to a total reaction volume of 50 µl to Table 2. Reaction components (reaction mix and template
successfully carry out primer extensions and other molecular DNA)
biology applications. Component Vol./reaction* Final concentration*
Taq 2x Master
Taq DNA Polymerase 2x Master Mix offers several advantages. 25 µl 1x
Mix
Set up time is significantly reduced. The chance of contaminating 25 mM MgCl2 X µl 1.5 mM (0.5 – 5 mM)
component stocks is eliminated. Reduction of reagent handling
Primer A (10 µM) 1 µl (0.5 – 5 µl) 0.2 µM (0.1 – 1.0 µM)
steps leads to better reproducibility. Standard tests can be set
up with the confidence that results will be consistent every time. Primer B (10 µM) 1 µl (0.5 – 5 µl) 0.2 µM (0.1 – 1.0 µM)
PCR-grade H2O X µl -
genomic DNA: 50 ng (10 – 500 ng)
Composition of the Taq DNA Polymerase 2x Master Mix (1.5 Template DNA X µl plasmid DNA: 0.5 ng (0.1 – 1 ng)
mM MgCl2 final concentration) bacterial DNA: 5 ng (1 – 10 ng)
TOTAL volume 50 µl -
 Tris-HCl pH 8.5, (NH4)2S04, 3 mM MgCl2, 0.2% Tween 20 * Suggested starting conditions; theoretically used conditions in brackets
 0.4 mM of each dNTP
 0.2 units/µl Ampliqon Taq DNA polymerase 3. Mix the reaction mix thoroughly and dispense appropriate
volumes into reaction tubes. Mix gently, e.g. by pipetting the
 Stabilizer
reaction mix up and down a few times.

Recommended Storage and Stability 4. Add template DNA to the individual tubes containing the
Long term storage at -20 °C. Product expiry at -20 °C is stated on reaction mix.
the label.
Option: Store at +4 °C for up to 6 months. 5. Program the thermal cycler according to the manufacturer´s
Quality Control instructions. See table 3 for an example.
Taq DNA Polymerase is tested for contaminating activities, with For maximum yield and specificity, temperatures and cycling
no traces of endonuclease activity, nicking activity or times should be optimized for each new template target or
exonuclease activity. primer pair.

Unit Definition 6. Place the tubes in the thermal cycler and start the reaction.
One unit is defined as the amount of polymerase that
incorporates 10 nmoles of dNTPs into acid-precipitable DNA in
30 minutes at 72 °C under standard assay conditions.
Table 3. Three-step PCR program Related Products
Cycles Duration of cycle Temperature
Taq Polymerase (500 units) * Cat. No.
1 2 – 5 minutes 95 °C
25 - 35 20 – 30 secondsa 95 °C Taq DNA Polymerase 5 U/µl A110003
20 – 40 secondsb 50 – 65 °C  with 10x Ammonium Buffer A111103
30 secondsc 72 °C  5x PCR Buffer RED A111803
1 5 minutesd 72 °C Taq DNA Polymerase 5 U/µl, RED A200003
a.  with 10x Ammonium Buffer A201103
Denaturation step: This step is the first regular cycling event and
consists of heating the reaction to 95 °C for 20 – 30 seconds. It causes Taq DNA Polymerase 5 U/µl, glycerol free A100003
melting of the DNA template by disrupting the hydrogen bonds  with 10x Ammonium Buffer A101103
between complementary bases, yielding single-stranded DNA
Hot Start Polymerase (500 units) * Cat. No.
molecules.
TEMPase Hot Start DNA Polymerase, 5 U/µl A220003
b.
Annealing step: The reaction temperature is lowered to 50 – 65 °C for  with 10x Ammonium Buffer A221103
20 – 40 seconds allowing annealing of the primers to the single-  5x PCR Buffer RED A221803
stranded DNA template. Typically, the annealing temperature is about
3 – 5 °C below the Tm (melting temperature) of the primers used. TEMPase Hot Start DNA Polymerase, glycerol free 5 U/µl A240003
c.
 with 10x Ammonium Buffer A241103
Extension/elongation step: Taq polymerase has its optimum activity
temperature at 72 °C. At this step the DNA polymerase synthesizes a High Fidelity - Proof reading (500 units) ** Cat. No.
new DNA strand complementary to the DNA template strand. The AccuPOL DNA Polymerase 2.5 U/µl A210003
extension time depends on the length of the DNA fragment to be  with 10x Ammonium Buffer A211103
amplified. As a rule of thumb, at its optimum temperature the DNA *Available in kits including one or two buffers (Ammonium Buffer, Standard Buffer
polymerase will polymerize a thousand bases per minute. or Combination Buffer). **AccuPOL only available in kits with Ammonium Buffer.
d. All kits include extra 25 mM MgCl2.
Final elongation: This single step is occasionally performed at a
temperature of 72 °C for 5 minutes after the last PCR cycle to ensure Buffers for DNA polymerases * Cat. No.
that a re ai i g si gle‐stra ded DNA is full e te ded. 10x Ammonium Buffer, 3 x 1.5 ml A301103
10x Standard Buffer, 3 x 1.5 ml A302103
10x Combination Buffer, 3 x 1.5 ml A303103
5x PCR Buffer RED, 6 x 1,5 ml ** A301810
*Ammonium Buffer, Standard Buffer and Combination Buffer are also available as
Mg2+ free buffers, detergent free buffers and Mg2+ and detergent free buffers.
**For direct gel loading and visualisation.

Taq Master Mixes (500 x 50 µl reactions) * Cat. No.

2x Master Mix, 1.5 mM MgCl2 final concentration A140303
2x Master Mix RED, 1.5 mM MgCl2 final concentration A180303
TEMPase Hot Start Master Mixes (500 x 50 µl reactions) * Cat. No.
2x Master Mix A**, 1.5 mM MgCl2 final concentration A230303
2x Master Mix A**BLUE, 1.5 mM MgCl2 final concentration A290403
*Master mixes available also in 1.1x variants as well as 2 mM MgCl2 variants, **Mix
A is Ammonium Buffer based, also available as Mix C based on Combination Buffer.

Special Master Mixes (500 x 50 µl reactions) Cat. No.

Multiplex 2x Master Mix, 3 mM MgCl2 final concentration A260303
GC TEMPase 2x Master Mix I – for GC-rich templates A331703
GC TEMPase 2x Master Mix II – for GC-rich templates A332703
Real-time PCR Master Mixes (400 x 25 µl reactions) Cat. No.
RealQ Plus 2x Master Mix for probe,
 without ROXTM A313402
 with low ROXTM A314402
 with high ROXTM A315402
RealQ Plus 2x Master Mix Green
 without ROXTM A323402
 with low ROXTM A324402
 with high ROXTM A325402
Ultrapure dNTPs* Cat. No.
dNTP Mix 40 mM (2 x 500 µl): 10 mM each dA, dC, dG, dT A502004
dNTP Set, 100 mM each: 250 µl of each dA, dC, dG and dT A511104
*Other concentrations and Single dNTPs are available.

Loading Buffers and Ladders Cat. No.

5x Loading Buffer Red *, 5 x 1 ml A608104
PCR DNA Ladder **, 100 – 3000 bp, 1 x 0.5 ml A610341
* Also available with Blue, Orange or Cyan. ** Available in different size ranges.

Reagents for in vitro laboratory use only.

Other product sizes, combinations and customized solutions are available. Please
look at www.ampliqon.com or ask for our complete product list for PCR Enzymes.
For customized solutions please contact us.
Made in Denmark

Issued 02/2017

Ampliqon A/S, Stenhuggervej 22, DK-5230 Odense M, Denmark. Phone: +45 70201169 Fax: +45 70201179 [email protected] www.ampliqon.com