Master mix for isothermal DNA amplification with EvaGreen and ROX
For in vitro use only!
Shipping: shipped on gel packs
Storage Conditions: store at -20 °C
Short term storage (up to 3 months) at 4 °C possible.
Shelf Life: 12 months
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
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.
Saphir Bst GreenMaster highROX (purple cap)
Saphier Bst Polymerase, dNTPs, reaction buffer, glycerol, EvaGreen DNA intercalator dye, ROX passive reference dye, stabilizers
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.
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.
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.
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||<500 ng/assay||x μl||x μl|
|PCR-grade Water||fill up to 20 μl||fill up to 50 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