Saphir RT-LAMP Kit

For general laboratory use.

Please centrifuge briefly before opening (volume ≤2 ml).

Shipping: shipped on gel packs

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
store dark

Shelf Life: 12 months

Form: liquid

Spectroscopic Properties: λ_exc 483 nm, λ_em 503 nm (dye bound to DNA)

Content:

Detection
Saphir RT-LAMP Kit synthesizes single-stranded DNA molecules (cDNA) complementary to the RNA template followed by loop-mediated isothermal DNA amplification. The kit additionally contains a green-fluorescent DNA stain that intercalates into DNA during the amplification process and allows the direct quantification of target RNA by fluorescence detection (analogous to real-time RT-PCR).

Assay design
Isothermal amplification is an extremely sensitive detection method and special care should be taken to avoid contamination of set-up areas and equipment with RNA/DNA of previous reactions.Preventing amplification in no-template controls due to carry-over contamination or amplification of unspecifically annealed primers or primer dimer formations can be challenging.

Primer design
Typically, 4 different primers are used to identify and amplify a distinct gene region 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 difficult 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 RNA/DNA preparation or analysis. No-template controls should be included in all amplifications.
First, prepare a 10 x conc. primer pre-mix. Second, set-up the isothermal amplification assay:

• 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)
• Select the FAM channel of the real-time instrument
• 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 "RNA/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