Saphir Bst Turbo GreenMaster

For general laboratory use.

Shipping: shipped on gel packs

Storage Conditions: store at -20 °C
store dark
Short term storage (up to 3 months) at 4 °C possible.

Shelf Life: 12 months

Form: liquid

Concentration: 2x conc.

Spectroscopic Properties: λ_exc 495 nm, λ_em 520 nm (dye bound to DNA)

Description:
Saphir Bst Turbo GreenMaster is a complete 2x conc. master mix for isothermal amplification of DNA. The mix is based on a genetically enhanced Bst polymerase of the next generation. The mixe is the ideal choice for ultra-fast and robust 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. The polymerase is 2-3x faster compared to Saphir Bst Polymerase (#PCR-389/#PCR-387) and allows detection of a target gene within 5-10 minutes.

Content:
Saphir Bst Turbo GreenMaster
Saphier Bst Turbo Polymerase, dNTPs, reaction buffer, glycerol, green-fluorescent DNA stain, additives and stabilizers

PCR-grade water

Detection
The mix contains a green-fluorescent DNA stain that intercalates into DNA during the amplification process and allows the direct quantification of target DNA by fluorescence detection (analogous to real-time PCR).
The mix can be combined with ROX reference dye (#PCR-351) to allow a signal normalization in real-time PCR instruments that are compatible with the evaluation of the ROX signal.

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 problem may be amplification in no-template controls due to carry-over contamination or amplification of unspecifically annealed primers or primer dimer formations.

Primer design
Typically, 4 different primers are used to identify 6 distinct DNA regions allowing the specific amplification of a target gene. An additional pair of primers further accelerates the amplification allowing to cut down the total detection time to 5-10 min.
The manual design of primers may be challenging due to the complex reaction sequence. To simplify the design process the use of a primer design software is recommended.
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
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.

Trouble shooting
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

Related products:

• Saphir Bst Turbo Polymerase, #PCR-390
• Green-Fluorescent DNA Stain, #PCR-378
• MgCl₂ Solution, #PCR-266
• dNTP Mix / 10 mM, #NU-1006
• dNTP Mix / 25 mM, #NU-1023"

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