Bst polymerase for isothermal DNA amplification
For in vitro use only!
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.
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.
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:
|component||stock conc.||final conc.||20 μl||50 μl|
|Saphir Bst Buffer||10x||1x||2 μl||5 μl|
|MgSO4 Stock Solution||25 mM||6-8 mM||3.2-4.8 μl||8.0-12.0 μl|
|dNTP Mix||10 mM||1.4 mM||2.8 μl||7 μl|
|Primer Mix||10x||1x||2 μl||5 μl|
|Saphir Bst Polymerase||8 units/μl||0.32 units/μl||0.8 μl||2 μl|
|EvaGreen DNA Stain||100 μM||1.3 μM||0.26 μl||0.65 μl|
|Template DNA||<500 ng/assay||x μl||x μl|
|PCR-grade Water||fill up to 20 μl||fill up to 50 μl|
Optimization of MgSO4 concentration:
A final Mg2+ concentration of 6.0 mM 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 volume||50 μl final assay volume|
|6 mM||3.2 μl||8.0 μl|
|7 mM||4.0 μl||10.0 μl|
|8 mM||4.8 μl||12.0 μl|
If amplification in no-template controls occurs the following points should be reviewed.
Cross contamination from environments
Carry-over contamination from previous reaction products
Non-template amplification from primers