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HOT START DNA POLYMERASES

ORDERING INFORMATION

HOT START DNA POLYMERASE (5 U/l)

Catalog #

Units

10x Reaction Buffer
50 mM MgCl2
5x Solution R (optional)

Price (EUR)

# HS 100
100 1 x 1.8 ml     
1.5 ml      
1 x 1.8 ml     

31,50

# HS 500
500

2 x 1.8 ml     

1.5 ml       2 x 1.8 ml     
126,00

RED HOT START DNA POLYMERASE (5 U/l)

Catalog #

Units

10x Reaction Buffer
50 mM MgCl2
5x Solution R (optional)

Price (EUR)

# Red-HS 100
100 1 x 1.8 ml     
1.5 ml      
1 x 1.8 ml     

38,50

# Red-HS 500
500

2 x 1.8 ml     

1.5 ml       2 x 1.8 ml     
150,00


DESCRIPTION

Hot Start DNA Polymerase is recommended for high specificity PCR reactions. The thermal activation prevents the extension of non-specifically annealed primers and primer-dimers formed at low temperatures during PCR set up.

Hot Start DNA Polymerase is a highly purified recombinant thermostable DNA polymerase that has been isolated from E. coli carrying a vector encoding the Thermus aquaticus DNA polymerase gene. The enzyme possesses a highly processive 53 DNA polymerase activity with optimum activity achieved at 74C. It exhibits high thermal stability in withstanding prolonged incubations at elevated temperatures (95C). Hot Start DNA Polymerase lacks 35 exonuclease activity.

Red Hot Start DNA Polymerase contains an inert red dye which allows identification of the reactions which contain enzyme. The dye has no adverse effect on PCR.

Activation step

Hot Start Polymerase requires no prolonged heating or denaturing step. The antibodies are quickly inactivated at the increased temperature of thermal cycling.

Unit Definition

One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmol of dNTP into acid-insoluble form in 30 minutes at 74C in the presence of the reaction buffer.

10x Reaction Buffer

670 mM Tris/HCl (pH 8.8 at 25C), 166 mM (NH4)2SO4, 4.5% Triton-X-100, 2 mg/ml gelatin.

Mg2+-Solution

50 mM MgCl2 (recommended final concentration: 1 4 mM).

Endodeoxyribonuclease Assay

No detectable conversion of covalently closed circular DNA to nicked DNA was observed after incubation of 10 units of Hot Start Taq DNA Polymerase with 1 g of digested DNA resp. 1l of pBR322 DNA in 50 l of Hot Start Taq Buffer with KCL containing 1.5 mM MgCl2 for 4 hours at 37C resp. 70C.

Exodeoxyribonuclease Assay

No detectable degradation of lambda DNA/HindIII fragments was observed after incubation of 10 units of Hot Start Taq DNA Polymerase with 1 g of digested DNA resp. 1 l of digested DNA in 50 l of Hot Start Taq Buffer with KCL containing 1.5 mM MgCl2 for 4 hours at 37C resp. 70C.

Ribonuclease Assay

No detectable degradation of 28S/18S bands was observed after incubation of 10 units of Hot Start Taq DNA Polymerase with 1 g of total RNA in 50 l of Hot Start Taq Buffer with KCL containing 1.5 mM MgCl2 for 4 hours at 37C resp. 70C.

Functional Assay

0.1 ng of lambda DNA was amplified using specific primers to produce a distinct 500 bp band.


BASIC PROTOCOL

Mix the following components on ice in a thin-walled 0.2 ml PCR-tube:

Component

Volume (in L)

Final concentration

10x Reaction Buffer

5

1x

50 mM MgCl2 Solution

1.5

1.5 mM

5 mM dNTP-Mastermix

2

200 M

Forward-Primer

variable

0.2 1 M

Reverse-Primer

variable

0.2 1 M

Template DNA

variable

< 1 g

Hot Start DNA Polymerase (5 U/l)

0.2 0.5

1 2.5 U

2x distilled, sterile water

Add to a final volume of 50


Total volume

50


To lower background we recommend the use of Solution R (10 l of 5x Solution R, final concentration 1x).


CYCLING PROGRAM

Step

Temperature (in C)

Time

Cycles

Initial activation

94

5 min

1

Denaturation

94

30 sec

23 35

Annealing

53*

45 sec

23 35

Extension

72

30 sec/kb

23 35

Final Extension

72

30 sec/kb

1

* : Approximately 5C below Tm of primers


HINTS AND NOTES

  • Mix the MgCl2 solution before use by vortexing vigorously.
  • Mix all components gently before use.
  • Mix the components of the reaction gently after pipetting. No vortexing, no centrifugation.
  • Keep the reaction tubes on ice as long as possible. Transfer the tubes from the ice to the cycler immediately after the denaturation temperature of about 94C has been reached.

Reaction conditions (incubation temperatures and times, concentrations of template DNA, primers, magnesium ions and enzyme) depend on template and primers used. Optimal MgCl2 concentrations vary between 1 4 mM and have to be determined empirically. However, many applications work at the standard concentration of 1.5 mM MgCl2. Advanced applications on genomic DNA require higher MgCl2 concentrations (2 3 mM) adjustable with the separate 50 mM MgCl2 solution supplied with the set.


OPTIMIZATION OF MgCl2 CONCENTRATION IN REACTION MIXTURE

Final concentration of MgCl2 in 50 l reaction volume

Add 50 mM MgCl2 solution

1.5 mM

1.5 l

1.75 mM

1.75 l

2.0 mM

2.0 l

2.5 mM

2.5 l

3.0 mM

3.0 l

4.0 mM

4.0 l


STORAGE

Store at -20C for 24 months.

WARNING

For research only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals.