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- Saphir - Model Bst2.0 - Polymerase
Saphir - Model Bst2.0 -Polymerase
Bst polymerase for isothermal DNA amplification. Isothermal Amplification. Saphir Bst2.0 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.
For in vitro use only!
- Shipping:shipped on blue ice
- Storage Conditions:store at -20 °C avoid freeze/thaw cycles
- Shelf Life:12 months
- Concentration:8 units/μl
Content:
Saphir Bst2.0 Polymerase (red cap)
8 units/μl Bst DNA Polymerase in 10 mM Tris-HCl, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1 % Triton X-100, 50 % (v/v) Glycerol, pH 7.5 (25 °C)
Saphir Bst2.0 Buffer (blue cap)
10 x conc. complete reaction buffer containing 200 mM Tris-HCl pH 8.8, 500 mM KCl, 100 mM (NH4)2SO4, 60 mM MgSO4, stabilizers and detergents
MgCl2 Stock Solution (yellow cap)
25 mM MgCl2
Detection
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 EvaGreen Fluorescent DNA Stain (#PCR-379) 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
- carry-over contamination or
- 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:
