5 U/μl, DNA-free ROTI®Pol Hot-TaqUltra, 40 µl, 1 x 40 µl User guide

1

ROTI®Pol Hot-TaqUltra

DNA-free recombinant hot start Taq DNA polymerase, antibody blocked,
for PCR amplifications, particularly of bacterial DNA
9350

1. Description

Hot start version of the recombinant full-length form of the heat stable Taq DNA polymerase from the
thermophilic bacterium Thermus aquaticus in storage buffer, and tested on the absence of bacterial
DNA.
For research use only. Not approved for use in clinical or in vitro diagnostics.

2. Applications

This ROTI®Pol Hot-TaqUltra is recommended for use in PCR applications in general, or if bacterial DNA,
or in RT-PCR, bacterial 16S rRNA shall be detected.
The polymerase is appropriate for use in the amplification of DNA from genomic, viral, and plasmid
templates, for high-yield PCR, for colony PCR and TA-cloning. The antibody-mediated blocking of the
DNA polymerase is released only at the initial denaturation step, hence resulting in highly specific
amplification of the target sequence without production of unwanted side products caused by unspecific
primer annealing.
ROTI®Pol Hot-TaqUltra is purified using a multiple-step process that minimises contaminating bacterial
DNA to a none detectable level. Each lot of the polymerase undergoes strict quality control testing in
order to ensure the absence of detectable amounts of contaminating bacterial DNA.
ROTI®Pol Hot-TaqUltra is able to amplify PCR products up to 3 kb with genomic DNA and up to at least
5 kb in size with Lambda DNA and is appropriate for use in the amplification of DNA from eukaryotic as
well as prokaryotic templates. The Hot-TaqUltra DNA polymerase possesses a 5´→ 3´ polymerase- as
well as a 5´-3’ exonuclease activity, and generates a 3’dA (adenine)-overhang which may well be used
for TA-cloning purposes.

3. Content

ROTI®Pol Hot-TaqUltra polymerase (Art. No. 9350) in storage buffer containing 50 % glycerol
Filled in red-capped tubes.

Reagent

Lid colour

9350.1

9350.2

Hot-TaqUltra polymerase

red

1 tube

5 tubes

This DNA polymerase is provided without reaction buffer.
Since the ROTI®Pol Hot-TaqUltra is an antibody-blocked, particularly high purified version of the TaqS

polymerase, the buffer included in the ROTI®Pol TaqS and Hot-TaqS products (as well as in the
ROTI®Pol TaqHY and Hot-TaqHY products) is perfectly suited for use as reaction buffer for the Hot­TaqUltra as well (see also 12. Additionally recommended products).
In addition to our specifically optimised reaction buffers, general Tris-based Taq polymerase reaction
buffers as, for instance, given in Molecular Cloning (Sambrook and Russell, Cold Spring Harbour
Laboratory Press), work well for the ROTI®Pol Hot-TaqUltra enzyme. We recommend use of 20 mM
MgCl2 and 0.1 % Tween®-20.

Please note, however, that the batches of our reaction buffers have not been tested for presence of
DNA or residual bacteria which might lead to false-positive results. For each buffer that shall be used for

the TaqUltra enzyme, either taken from a ROTI®Pol product or self-made, we recommend to do
preliminary studies in order to ensure the freeness of DNA functioning as target for the particular primer
pairs to be used.

Instructions for use

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4. Storage Buffer

50 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.1 mM EDTA, 0.5 % IGEPAL CA-630, 0.5 % Tween-20,
1 mM DTT, 50 % glycerol, mouse anti-Taq IgG

5. Enzyme activity

5 units/µl enzyme solution

6. Unit definition

One unit of activity is defined as the amount of enzyme required to incorporate 10 nmoles of dNTP into
an acid-insoluble DNA fraction in 30 minutes at 72 °C.

7. Suggested pipetting scheme

Due to the inhibition of polymerase activity at room temperature all reactions may be set up at room
temperature. This will not result in an increase of unspecific product or primer-dimer formation.

Components

Apply for PCR reaction
of 20 μl volume

Final concentration
(recommended)

PCR buffer (10x)*

2 µl

1x

dNTP-Mix (2 mM)

2 µl

800 μM (200 μM each)

Forward primer (e.g. 5 pmol/µl)

variable (e.g. 1 µl)

0.1-0.5 µM

Reverse primer (e.g. 5 pmol/µl)

variable (e.g. 1 µl)

0.1-0.5 µM

Template DNA

variable

0.01-10 ng / reaction

ROTI®Pol Hot-TaqUltra polymerase (5 U/µl)

variable (i.e. 0.2 µl)

0.5-1.5 U

Sterile dest. water

adjust to 20 μl final volume

*also see 3. Content

8. Basic amplification protocol

Step

Time

Temperature

Initial denaturation

2 minutes

92-95 °C

25-40 cycles

Denaturation

2-10 seconds

92-95 °C

Annealing

2-10 seconds

55-68 °C

Extension

variable, depends on the length of product

72 °C

9. Notes

IMPORTANT: For specific species-DNA detection and to avoid false positive results make sure your

PCR buffer and other PCR reagents are free of DNA contaminations.
For maximum yield and specificity, annealing temperatures and annealing time as well as extension time

and cycle numbers should be optimised for each template target and primer pair. Usually the optimal
annealing temperature is 2-5 °C below the melting temperature of the primers. Recommended
elongation time is 30-60 secs. per 1 kb of target. Elongation times of 30 secs. per 1 kb may be sufficient
but longer elongation times may be necessary depending on the complexity of the template DNA.

Product is not covered by pending or issued patents or may have certain limitations. To our best
knowledge, however, this product does not provide any conflict with pending or issued patents.

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10. Recommended MgCl2 concentration

2-4 mM
In case the MgCl2 concentration has to be adjusted, use a separate MgCl2 solution (100 mM) in PCR
quality and add in appropriate amounts according to the scheme below. We recommend doing PCR with
a MgCl2 gradient in order to find the optimal concentration.

Pipetting scheme for additional MgCl2

Final MgCl2 conc. in mM

2.5 3 3.5

4

Add 100 mM MgCl2 solution in following
amounts to 20 μl reaction volume

0.1 µl

0.2 µl

0.3 µl

0.4 µl

11. Storage conditions

Store at -20 oC. Infrequent short term storage (few hours) of the enzyme may be done at +4 °C.

12. Additionally recommended products
For our Thermal cyclers please contact us under 0721 / 5606 - 0

ROTI®Mix PCR 3 (10 mM per dNTP dATP, dTTP, dGTP, dCTP) Art. No. L785
ROTI®Mix PCR 3 (pH 7) (10 mM per dNTP dATP, dTTP, dGTP, dCTP) Art. No. 0179
dNTP-Set 1 (≥99 %, 100 mM pure solutions dATP, dTTP, dGTP, dCTP) Art. No. K039
dNTP-Set 1 (pH 7) (≥99 %, 100 mM pure solutions dATP, dTTP, dGTP, dCTP) Art. No. 0178

PCR water for molecular biology, sterile, ready-to-use Art. No. 1HPE
Magnesium chloride solution 25 mM, for PCR, for molecular biology Art. No. 1HY7
Mineral oil (for or overlaying PCR and other enzymatic reactions) Art. No. HP50

ROTI®Nucleic acid-free (ready-to-use solution for removal of DNA from surfaces) Art. No. HP69
ROTI®Nucleic acid-free eXtra (ready-to-use, gentle solution for DNA removal) Art. No. 1312
DNA AWAY

®

(ready-to-use solution for removal of DNA from surfaces) Art. No. X996

Please note our full range of DNA polymerases and MasterMixes:

ROTI®Pol TaqS Art. No. 9223
ROTI®Pol TaqS Mix Art. No. 9239
ROTI®Pol TaqS Red-Mix Art. No. 9241
ROTI®Pol Hot-TaqS Art. No. 9245
ROTI®Pol Hot-TaqS Mix Art. No. 9248
ROTI®Pol Hot-TaqS Red-Mix Art. No. 9256
ROTI®Pol TaqHY Art. No. 9345
ROTI®Pol TaqHY Mix Art. No. 1K33
ROTI®Pol TaqHY Red-Mix Art. No. 1K34
ROTI®Pol Hot-TaqHY Art. No. 9346
ROTI®Pol ProofRead Art. No. 9344
ROTI®Pol TaqUltra Art. No. 9347
ROTI®Pol Hot-TaqUltra Art. No. 9350

ROTI

®

Pol Hot-TaqUltra 200 U 9350.1

1.000 U 9350.2

Carl Roth GmbH + Co. KG

Schoemperlenstraße 3-5 • 76185 Karlsruhe • P.O. Box 100121 • 76231 Karlsruhe
Phone: [phone] • Fax: [phone] • [e-mail] • www.carlroth.com

The company is a limited partnership with headquarters in Karlsruhe, reg. court Mannheim HRA 100055. Roth Chemie GmbH, with headquarters in Karlsruhe,
reg. court Mannheim HRB 100428, is the personally liable partner. Managing Director: André Houdelet. Sales tax identification number: DE 143621073. jh 01/2022