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Saphir Bst2.0 GreenMaster highROX

Master mix for isothermal DNA amplification with EvaGreen and ROX

Isothermal Amplification

Cat. No. Amount Price (EUR) Buy / Note
PCR-388S 2 x 1,25 ml 89,70 Add to Basket/Quote Add to Notepad
PCR-388L 10 x 1,25 ml 359,00 Add to Basket/Quote Add to Notepad

For in vitro use only!

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

Description:
Saphir Bst2.0 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.

Content:
Saphir Bst2.0 GreenMaster highROX (purple cap)
Saphier Bst2.0 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:

componentstock conc.final conc.20 μl50 μl
Saphir Bst2.0 GreenMaster highROX2x1x10 μl25 μl
Primer Mix10x1x2 μl5 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.


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

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