Saphir Bst GreenMaster highROX

Master mix for isothermal DNA amplification with EvaGreen and ROX

Isothermal Amplification

Catálogo Nº Apresentação Preço (R$) Comprar
PCR-388S 2 x 1,25 mlSob demanda Adicionar ao Carrinho
PCR-388L 10 x 1,25 mlSob demanda Adicionar ao Carrinho

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: EvaGreen bound to DNA: λexc 500 nm, λem 530 nm ROX passive reference dye: λexc 576 nm, λem 601 nm

Descrição:
Saphir Bst GreenMaster highROX 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 10 9 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 highROX (purple cap)
Saphier Bst Polymerase, dNTPs, reaction buffer, glycerol, EvaGreen DNA intercalator dye, ROX passive reference dye, stabilizers

PCR-grade water

Detection
The mix contains the fluorescent DNA stain EvaGreen® 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 contains 500 nM ROX passive reference dye in the final assay. The dye does not take part in the PCR reaction but allows to normalize for non-PCR related signal variation and provides a baseline in multiplex reactions.

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 highROX||2x||1x||10 μl||25 μl
Primer Mix||10x||1x||2 μl||5 μl
Template DNA||||

Produtos relacionados:

  • Saphir Bst Polymerase, #PCR-389
  • EvaGreen DNA Stain, #PCR-379
  • MgCl2 Stock Solution, #PCR-266
  • dNTP Mix / 10 mM, #NU-1006
  • dNTP Mix / 25 mM, #NU-1023