RT-LAMP Colorimetric Kit - lyo ready

Name: RT-LAMP Colorimetric Kit - lyo ready
Brand: Jena Bioscience GmbH
SKU: PCR-541S

PCR-541S

Kit for fast isothermal RNA amplification with visible colorimetric readout
Isothermal Amplification

-30 %

209,10 €

Amount

Add to cart Add to quote Save for later

For research use only.
Please centrifuge briefly before opening (volume ≤2 ml).

Description:
RT-LAMP Colorimetric Kit – lyo ready is an optimised formulation containing a visible pH depending dye for rapid and easy detection of the LAMP reaction.
This kit allows a fast, visual detection of the amplification by a clear pink-to-yellow color change.

Lyophilisation
RT-LAMP Colorimetric Kit – lyo ready is recommended for preparation of lyophilised RT-LAMP assays.
The additive mix contains cryoprotectants and lyoprotectants for optimal stability and structure formation.

Shipping: shipped on gel packs

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

Shelf Life: 12 months

Form: liquid

Content:

Component	Cap	PCR-541S	PCR-541L
RT-LAMP Buffer	yellow	800 μl	5 x 800 μl
RT-LAMP Enzyme Mix	red	240 μl	5 x 240 μl
RT-LAMP Additive Mix	violet	1.6 ml	5 x 1.6 ml
PCR-grade Water	white	1.2 ml	3 x 1.2 ml

Detection
The ongoing amplification can be observed directly with the naked eye by a color change of the assay. The color changes from pink to yellow as the amount of amplified DNA increases. Depending on the amount of template material used, the reaction is completed after 20 to 30 minutes.
The change depends on the pH change of the reaction system. Make sure not to inhibit this change by template DNA or RNA stored in Tris-buffer containing nucleic acid storage solution. PCR-grade Water is recommended to store RNA, DNA and primers.

Assay design
Isothermal amplification is an extremely sensitive detection method and care should be taken to avoid contamination of setup areas and equipment with DNA of previous reactions. A common problem is amplification in no-template controls due to

1. carry-over contamination or

2. amplification of unspecifically annealed primers or
primer dimer formations.

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
Depending on the setup used, a reaction volume of 20-50 μl is recommended for most applications. Pipette with sterile filter tips and perform the setup in a separate area from the DNA preparation or analysis. No-template controls should be included with all amplifications.

First, prepare a 10x conc. primer premix as follows:

primer	stock conc.	final conc.	100 μl	1 ml
FIP	100 μM	16 μM	16 μl	160 μl
BIP	100 μM	16 μM	16 μl	160 μl
F3	100 μM	2 μM	2 μl	20 μl
B3	100 μM	2 μM	2 μl	20 μl
Loop F	100 μM	4 μM	4 μl	40 μl
Loop B	100 μM	4 μM	4 μl	40 μl
PCR-grade Water			56 μl	560 μl

Set up for a liquid isothermal amplification assay
Prepare a master mix as follows:

component	stock conc.	final conc.	20 μl	50 μl
RT-LAMP Buffer	5 x	1 x	4 μl	10 μl
RT-LAMP Enzyme Mix	18 x	1 x	1.1 μl	2.8 μl
Primer Mix	10 x	1 x	2 μl	5 μl
Template RNA		<100 ng/assay	x μl	x μl
PCR-grade Water			fill up to 20 μl	fill up to 50 μl

Set up for a lyo-ready isothermal amplification assay for freeze-drying
Prepare a master mix as follows:

component	stock conc.	final conc.	20 μl	50 μl
RT-LAMP Buffer	5 x	1 x	4 μl	10 μl
RT-LAMP Enzyme Mix	18 x	1 x	1.1 μl	2.8 μl
Primer Mix¹	10 x	1 x	2 μl	5 μl
RT-LAMP Additive Mix	2.5 x	1 x	8 μl	20 μl
PCR-grade Water			fill up to 20 μl	fill up to 50 μl

¹ optional, if primers should be included in the lyophilised mix

Dispensing the master mix:
Vortex the mix prepared above thoroughly to assure homogeneity. Dispense the appropriate volume to each PCR tube or well of the plate.

Lyophilisation of the master mix:
Use a freeze dryer or sublimator to freeze-dry the prepared master mix. Follow the instructions provided by the freeze-dryer manufacturer.

Final assay preparation:
To prepare the final liquid assay, rehydrate the lyophilisate with primers (if not already included in the lyophilisate) and template RNA to obtain the final assay volume. Do not exceed 100 ng RNA per assay. Add PCR-grade Water for no-template controls. Close or seal the tubes/plates and centrifuge briefly to remove bubbles.

Amplification and detection

Use a thermoblock or PCR cycler to run the assays
Set the instrument to a constant incubation temperature between 60 - 65°C (depending on the primer annealing temperature)
Check the visible color change in appropriate intervals for up to 30 min.

Trouble shooting
If amplification in no-template controls occurs the following points should be reviewed.

The pink-to-yellow color change depends on the pH change of the reaction system. Make sure not to inhibit this change by template RNA stored in Tris-buffer containing nucleic acid storage solution. PCR-grade Water is recommended to store RNA, DNA and primers.

When using the product as a liquid mix (non‑lyophilized), the initial color may appear dark orange rather than pink. This is acceptable and does not indicate reduced performance; the expected color shift will still proceed towards yellow.

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