Taq DNA Polymerase 2x Master Mix RED Protocol

1.5 mM MgCl2 final concentration This protocol serves as a guideline to ensure optimal PCR results
when using Taq DNA Polymerase 2x Master Mix RED. Optimal
reaction conditions such as incubation times, temperatures, and
Cat. No.: A180306 amount of template DNA may vary and must be determined
individually.
2500 Reactions
Taq DNA Polymerase 2x Master Mix RED, 1.5 mM
-
MgCl2 1. Thaw Taq 2x Master Mix RED and primers. It is important to
ID No. 5200300 thaw the solutions completely and mix thoroughly before
Cap colour Red use to avoid localized concentrations of salts. Keep all
Content 50 x 1.25 ml components on ice.

2. Prepare a reaction mix. Table 1 shows the reaction set up for

a final volume of 50 L. If desired, the reaction size may be
scaled down. Use 10 µl of the Taq 2x Master Mix RED in a
final volume of 20 µl.

Table 1. Reaction components (reaction mix and template

DNA)
Key Features Component Vol./reaction* Final concentration*
Taq 2x Master
25 µl 1x
Taq DNA Polymerase 2x Master Mix RED is a ready-to-use 2x Mix
+
reaction mix with the Ampliqon Taq DNA polymerase, the NH4 25 mM MgCl2 0 µl (0 – 6 µl) 1.5 mM (1.5 – 4.5 mM)
buffer system, dNTPs and magnesium chloride present. Each Primer A (10 µM) 1 µl (0.5 – 5 µl) 0.2 µM (0.1 – 1.0 µM)
reaction requires 25 µl of the 2x Master Mix RED. Simply add Primer B (10 µM) 1 µl (0.5 – 5 µl) 0.2 µM (0.1 – 1.0 µM)
primers, template and water to a total reaction volume of 50 µl
PCR-grade H2O X µl -
to successfully carry out primer extensions and other molecular
genomic DNA: 50 ng (10 – 500 ng)
biology applications.
Template DNA X µl plasmid DNA: 0.5 ng (0.1 – 1 ng)
Taq DNA Polymerase 2x Master Mix RED offers several bacterial DNA: 5 ng (1 – 10 ng)
advantages. Set up time is significantly reduced. The chance of
contaminating component stocks is eliminated. Reduction of TOTAL volume 50 µl -
* Suggested starting conditions; theoretically used conditions in brackets
reagent handling steps leads to better reproducibility. Standard
tests can be set up with the confidence that results will be
3. Mix the reaction mix thoroughly and dispense appropriate
consistent every time.
volumes into reaction tubes. Mix gently, e.g. by pipetting the
There is no need to buy and use separate loading dyes. Simply
reaction mix up and down a few times.
load a portion of the reaction product onto an agarose gel for
4. Add template DNA to the individual tubes containing the
electrophoresis and subsequent visualization. The red dye front
reaction mix.
runs at 1000 – 2000 bp on a 0.5 – 1.5 % agarose gel.
5. Program the thermal cycler according to the manufacturer´s
instructions. See table 2 for an example.
Composition of the Taq DNA Polymerase 2x Master Mix RED
For maximum yield and specificity, temperatures and cycling
(1.5 mM MgCl2 final concentration)
times should be optimized for each new template target or
primer pair.
 Tris-HCl pH 8.5, (NH4)2S04, 3 mM MgCl2, 0.2% Tween 20
 0.4 mM of each dNTP 6. Place the tubes in the thermal cycler and start the reaction.
 Ampliqon Taq DNA polymerase
 Inert red dye and stabilizer
7. At the end of the run, simply load a portion of the reaction
product (e.g. 10 µl) onto an agarose gel for analysis.
Recommended Storage and Stability
Table 2. Three-step PCR program
Long term storage at -20 °C. Product expiry at -20 °C is stated on
Cycles Duration of cycle Temperature
the label. 1 2 – 5 minutes 95 °C
Option: Store at +4 °C for up to 6 months. 25 - 35 20 – 30 secondsa 95 °C
20 – 40 secondsb 50 – 65 °C
Quality Control 30 secondsc 72 °C
Taq DNA Polymerase is tested for contaminating activities, with 1 5 minutesd 72 °C
no traces of endonuclease activity, nicking activity or a.
Denaturation step: This step is the first regular cycling event and
exonuclease activity. consists of heating the reaction to 95 °C for 20 – 30 seconds. It causes
melting of the DNA template by disrupting the hydrogen bonds
between complementary bases, yielding single-stranded DNA
molecules.
b.
Annealing step: The reaction temperature is lowered to 50 – 65 °C for
20 – 40 seconds allowing annealing of the primers to the single-
stranded DNA template. Typically, the annealing temperature is about
3 – 5 °C below the Tm (melting temperature) of the primers used.
c.
Extension/elongation step: Taq polymerase has its optimum activity
temperature at 72 °C. At this step the DNA polymerase synthesizes a
 new DNA strand complementary to the DNA template strand. The Related Products
extension time depends on the length of the DNA fragment to be
amplified. As a rule of thumb, at its optimum temperature the DNA Taq Master Mixes (500 x 50 µl reactions) * Cat. No.
polymerase will polymerize a thousand bases per minute. 2x Master Mix, 1.5 mM MgCl2 final concentration A140303
d.
Final elongation: This single step is occasionally performed at a 2x Taq OptiMix CLEAR, 1.5 mM MgCl2 final concentration A370503
temperature of 72 °C for 5 minutes after the last PCR cycle to ensure
2x Master Mix RED, 1.5 mM MgCl2 final concentration A180303
that any remaining single‐stranded DNA is fully extended.
TEMPase Hot Start Master Mixes (500 x 50 µl reactions) * Cat. No.
Two-step PCR program
2x Master Mix A**, 1.5 mM MgCl2 final concentration A230303
Fast 2-step PCR protocols are available using this link: 2x Master Mix A**BLUE, 1.5 mM MgCl2 final concentration A290403
https://ampliqon.com/en/pcr-technology/application-notes/ *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 TEMPase Master Mixes (500 x 50 µl reactions) Cat. No.
Notes: Multiplex 2x Master Mix, 3 mM MgCl2 final concentration A260303
 The final MgCl2 concentration of this 2x Taq Master Mix RED GC TEMPase 2x Master Mix I – for GC-rich templates A331703
is 1.5 mM. In some applications, more than 1.5 mM MgCl2 is GC TEMPase 2x Master Mix II – for GC-rich templates A332703
2+
required for best results. Use 25 mM to adjust the Mg
concentration according to table 3. Taq DNA Polymerase (500 units) * Cat. No.
Taq DNA Polymerase 5 U/µl A110003
Table 3. Additional volume (µl) of MgCl2 per 50 µl reaction:  with 10x Ammonium Buffer A111103
Final MgCl2 conc. *Available in kits including one or two buffers (Ammonium Buffer, Standard Buffer
1.5 2.0 2.5 3.0 3.5 4.0 4.5 or Combination Buffer). All kits include extra 25 mM MgCl2
in reaction (mM)
Hot Start DNA Polymerase (500 units) * Cat. No.
Volume of 25 mM MgCl2 0 1 2 3 4 5 6
TEMPase Hot Start DNA Polymerase, 5 U/µl A220003
 with 10x Ammonium Buffer A221103
*Available in kits including one or two buffers (Ammonium Buffer, Standard Buffer
or Combination Buffer). All kits include extra 25 mM MgCl2

Buffers for DNA polymerases * Cat. No.

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
PCR Grade Water, 6 x 5 ml A360056
*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.

For Research Use Only. Not for use in diagnostics procedures.

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 08/2021

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