Saphir Bst GreenMaster

For general laboratory use.

Envio: shipped on gel packs

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

Validade: 12 months

Forma: liquid

Concentração: 2x conc.

Propriedades espectroscópicas: λexc 495 nm (SYBR®Green bound to DNA), λem 520 nm (SYBR®Green bound to DNA)

Descrição:
Saphir Bst GreenMaster is a complete 2x conc. master mix for isothermal amplification of DNA. The mix is based on a genetically optimized Bst polymerase that allows 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. LAMP technique allows detection of a target gene within 10 - 30 minutes.

Contente:
Saphir Bst GreenMaster (orange cap)
Saphier Bst Polymerase, dNTPs, reaction buffer, glycerol, SYBR®Green DNA intercalator dye, stabilizers

PCR-grade water

Detection
The mix contains the fluorescent DNA stain SYBR®Green 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 10-20 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:
component||stock conc.||final conc.||20 μl||50 μl
Saphir Bst GreenMaster||2x||1x||10 μl||25 μl
Primer Mix||10x||1x||2 μl||5 μl
Template DNA||||

Produtos relacionados:

• Saphir Bst Polymerase, #PCR-389
• SYBR®Green Fluorescent DNA Stain, #PCR-378
• MgCl2 Stock Solution, #PCR-266
• dNTP Mix / 10 mM, #NU-1006
• dNTP Mix / 25 mM, #NU-1023