Saphir Bst2.0 Polymerase

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

Description:
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.

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 SybrGreen or EvaGreen Fluorescent DNA Stain (#PCR-378, #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
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:

• 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 MgCl₂ concentration:
A final Mg²⁺ concentration of 6.0 mM (as contained in the reaction buffer) is optimal for most primer-template combinations. However, if an individual Mg²⁺ optimization is essential add 25 mM MgCl₂ stock solution (#PCR-266) as shown in the table below.

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
• Use a master mix with UNG and dUTP instead of dTTP or add both components separately to the assay to prevent carry-over contamination

Non-template amplification from primers

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

Related products:

• SybrGreen DNA Stain, #PCR-378
• EvaGreen DNA Stain, #PCR-379
• MgCl₂ Stock Solution, #PCR-266
• dNTP Mix / 10 mM, #NU-1006
• dNTP Mix / 25 mM, #NU-1023