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Saphir Bst Polymerase

Bst polymerase for isothermal DNA amplification

Isothermal Amplification

Cat. No. Amount Price (EUR) Buy / Note
PCR-380S 2.000 Units 79,00 Add to Basket/Quote Add to Notepad
PCR-380L 10.000 Units 316,00 Add to Basket/Quote Add to Notepad

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 Bst Polymerase is an 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 Bst 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 Bst Buffer
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:

componentstock conc.final conc.20 μl50 μl
Saphir Bst Buffer10x1x2 μl5 μl
MgCl2 Stock Solution25 mM0-2 mM0-1.6 μl0-4 μl
dNTP Mix10 mM400 μM0.8 μl2 μl
Primer Mix10x1x2 μl5 μl
Saphir Bst Polymerase8 units/μl0.32 units/μl0.8 μl2 μl
SybrGreen or
EvaGreen DNA Stain
100 μM1.5 μM

2.5 mM
0.3 μl

0.5 μl
0.75 μl

1.25 μl
Template DNA<500 ng/assayx μlx μl
PCR-grade Waterfill up to 20 μlfill up to 50 μl

  • 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.

final MgCl2 conc.20 μl final assay volume50 μl final assay volume
6 mM--
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
  • 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

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