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Saphir Bst Polymerase

Bst polymerase for isothermal DNA amplification

Isothermal Amplification

Cat. No. Amount Price (EUR) Buy / Note
PCR-389S 2.000 Units98,10 Add to Basket/Quote Add to Notepad
PCR-389L 5 x 2.000 Units392,50 Add to Basket/Quote Add to Notepad

For general laboratory use.

Shipping: shipped on gel packs

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months

Concentration: 8 units/μl

Saphir Bst Polymerase is a genetically improved Bst polymerase for rapid and specific amplification of DNA at constant temperature (60 to 65 °C). The enzyme shows high strand displacement activity and generates an amplification factor of up to 109 which is comparable to approx. 30 cycles in a PCR assay. This allows detection of a target gene within 10-30 minutes.

Saphir Bst Polymerase
8 units/μl Bst DNA Polymerase in 10 mM Tris-HCl, 50 mM KCl, 50 % (v/v) Glycerol, pH 7.5 (25 °C) and stabilizers.

Saphir Bst Buffer
10 x conc. complete reaction buffer containing 200 mM Tris-HCl pH 8.8, KCl, (NH4)2SO4, 20 mM MgSO4 and detergents.

MgSO4 Stock Solution
25 mM MgSO4.

Although some methods have been developed to visualize DNA amplification by basic equipment or even the naked eye (increase of turbidity, color change of added dyes, hybridization to gold-bound ss-DNA) in general real-time detection of the DNA amplification by a fluorescent DNA-intercalator dye is recommended. Addition of a Fluorescent DNA Stain to the assay allows a sensitive measurement of the increasing amount of DNA without influence on the reaction.

Assay design
Isothermal amplification is an extremely sensitive detection method and care should be taken to avoid contamination of set-up areas and equipment with DNA of previous reactions. A common problem is amplification in no-template controls due to
1. carry-over contamination or
2. amplification of unspecifically annealed primers or primer dimer formations.
As sensitivity and non-template amplification of in-silico designed primers may vary, the evaluation of 2-4 real primer sets before choosing a final set is recommended.

Assay set-up
Depending on the detection method and machine a reaction volume of 20-50 μl is recommended for most applications. Pipet with sterile filter tips and perform the set-up in an area separate from DNA preparation or analysis. No-template controls should be included in all amplifications.
First, prepare a 10x conc. primer pre-mix. Second, set-up the isothermal amplification assay:

componentstock conc.final conc.20 μl50 μl
Saphir Bst Buffer10x1x2 μl5 μl
MgSO4 Stock Solution *25 mM0-2 mM0-1.6 μl0-4 μl
dNTP Mix10 mM1.4 mM2.8 μl7 μl
Primer Mix10x1x2 μl5 μl
Saphir Bst Polymerase8 units/μl0.32 units/μl0.8 μl2 μl
EvaGreen DNA Stain (#PCR-379)100 μM1.3 μM0.26 μl0.65 μl
Template DNA<500 ng/assayx μlx μl
PCR-grade Waterfill up to 20 μlfill up to 50 μl

* optional, please refer to the table below

  • Use a specific detection instrument for isothermal amplification or a real-time PCR cycler to run the assays
  • Set the instrument to a constant incubation temperature between 60 to 65°C (depending on the primer annealing temperature)
  • Measure the fluorescence intensity at an interval of 1 min for up to 30 min.

Optimization of MgSO4 concentration:
A final Mg2+ concentration of 6.0 mM (as already contained in the reaction buffer) is optimal for most primer-template combinations. However, if an individual Mg2+ optimization is essential add 25 mM MgSO4 stock solution as shown in the table below.

final MgSO4 conc.20 μl final assay volume50 μl final assay volume
6 mM- μl- μl
7 mM0.8 μl2.0 μl
8 mM1.6 μl4.0 μl

Trouble shouting
If amplification in no-template controls occurs the following points should be reviewed.

Cross contamination from environments

  • Clean equipment and areas with “DNA Away” solution
  • Replace reagent stocks and pre-mixes with new components
  • Stop reactions at an earlier point of time before non-template amplification occur

Carry-over contamination from previous reaction products

  • Avoid opening reaction vessels after amplification
  • Use separate preparation area and equipment if post-reaction processing is necessary

Non-template amplification from primers

  • Increase incubation temperature stepwise by 1-2 °C
  • Design a new set of primers for the target sequence

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