HOT START DNA POLYMERASES
ORDERING INFORMATION
HOT START DNA POLYMERASE (5 U/µl)
|
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 |
|
2 x 1.8 ml |
1.5 ml | 2 x 1.8 ml |
126,00 |
RED HOT START DNA POLYMERASE (5 U/µl)
|
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 |
|
2 x 1.8 ml |
1.5 ml | 2 x 1.8 ml |
150,00 |
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 5’→3’ DNA polymerase activity with optimum activity achieved at 74°C. It exhibits high thermal stability in withstanding prolonged incubations at elevated temperatures (95°C). Hot Start DNA Polymerase lacks 3’→5’ exonuclease activity.
Activation step
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 74°C in the presence of the reaction buffer.
10x
Reaction Buffer
670 mM Tris/HCl (pH 8.8 at 25°C), 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
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 µg of digested DNA resp. 1 µl of digested DNA in 50
µl of Hot Start Taq Buffer with KCL containing 1.5 mM MgCl2 for 4 hours at 37°C
resp. 70°C.
Ribonuclease
Assay
Functional
Assay
BASIC PROTOCOL
Mix the following components on ice in a thin-walled 0.2 ml PCR-tube:
|
Component |
Volume (in µL) |
Final concentration |
|
10x Reaction
Buffer |
5 |
1x |
|
50 mM MgCl2
Solution |
1.5 |
1.5 mM |
|
5 mM
dNTP-Mastermix |
2 |
200 µM |
|
Forward-Primer |
variable |
0.2 – 1 µM |
|
Reverse-Primer |
variable |
0.2 – 1 µM |
|
Template DNA |
variable |
< 1 µg |
|
Hot Start DNA
Polymerase (5 U/µl) |
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 µl 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 5°C 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 94°C 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 µl reaction volume |
Add 50 mM MgCl2 solution |
|
1.5 mM |
1.5 µl |
|
1.75 mM |
1.75 µl |
|
2.0 mM |
2.0 µl |
|
2.5 mM |
2.5 µl |
|
3.0 mM |
3.0 µl |
|
4.0 mM |
4.0 µl |
STORAGE
Store at -20°C 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.
