Lyophilisate for isothermal DNA amplification with green-fluorescent DNA stain
For general laboratory use.
Shipping: shipped at ambient temperature
Storage Conditions: store at ambient temperature
Store in an aluminium-coated bag or in a dry place.
Lyophilisates may hydrate at humidity levels >70 % when sealing is opened.
Shelf Life: 6 months
Saphir Bst GreenMaster Lyophilisate is a freeze-dried master mix for isothermal amplification of DNA using LAMP (Loop-mediated Isothermal Amplification). The mix is based on the activity of Bst polymerase and is the ideal choice for 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.
Saphir Bst GreenMaster Lyophilisate allows detection of a target gene within 10-20 minutes.
Saphir Bst GreenMaster Lyophilisate
Saphir Bst Polymerase, dNTPs, reaction buffer, Green DNA intercalator dye, additives and stabilizers in freeze-dried format for 20 μl final assay volume
PCR grade water
Saphir Bst GreenMaster Lyophilisate is delivered in 8-tube strips or 96-well plates preloaded with a complete master mix in a dry, room temperature stable format. The lyophilisate combines highest performance with convenience of use and stability. There is no need for freezing, thawing or pipetting on ice. The few remaining pipetting steps minimize the risk of errors or contaminations.
Each vial contains all components (except primers and template) required for a 20 μl LAMP assay.
To perform the assay, only fill up the vials with a primer mix and add DNA template.
The lyophilisate can also be used with ROX reference dye in PCR instruments that are compatible with the evaluation of the ROX signal. In this case, the ROX dye (#PCR-351) should be added as 1x concentration to the PCR reaction.
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.
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.||Volumne 1x 20 μl Assay|
|Primer Mix||10x||1x||2 μl|
|Template DNA||<500 ng/assay||x μl|
|PCR-grade Water||fill up to 20 μl|
Dispensing the master mix
Vortex the primer/probe mix thoroughly to assure homogeneity.
Dispense 20 μl to each PCR tube or well of the plate.
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
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