[NIPPON GENE] Nucleic Acid Amplification Reagents for LAMP
LAMP MASTER Series are master mix reagents for isothermal nucleic acid amplification by LAMP. Because 2 x LAMP MASTER contains thermostable strand displacement DNA polymerase, Mg2+, dNTPs, and the optimized buffer necessary for LAMP, DNA can be amplified by LAMP on adding primers and template nucleic acid, alone.
Reagent selection according to detection method
Turbidity detection [LAMP MASTER for Turbidity]
When 2 x LAMP MASTER is used alone, the turbidity of the reaction solution can be measured to detect amplification.
Fluorescence detection [LAMP MASTER for Fluorescence]
Amplification can be detected with a real-time PCR device by adding the intercalator provided with the set product.
Visual determination [LAMP MASTER for Turbidity (Visible Dye)]
Amplification can be visually confirmed by adding the fluorescent visual reagent provided with the set product.
Example of use
RT-LAMP (One Step) with the use of LAMP MASTER for Fluorescence
Total RNA was extracted from HeLa cells using ISOSPIN Cell & Tissue RNA (Product Number: 314-08211) and then used as template RNA. The process, from amplification to detection, was carried out in one step using a real-time PCR device by additionally mixing AMV Reverse Transcriptase (Product Number: 311-07501), with 2 x LAMP MASTER, 10 x Intercalation Mix, and a primer set (target region: GAPDH) to prepare a reaction solution.
Reaction conditions
| 2 x LAMP MASTER | 12.5 µL |
| 10 x Intercalation Mix | 2.5 µL |
| 10 x LAMP Primer Mix | 2.5 µL |
| AMV Reverse Transcriptase | 0.2 units (1 µL)※ |
| Template RNA | 0.5, 5, 50 ng (1 µL) |
| ddWater | up to 25 µL |
| 68℃ 60 min → Melting curve analysis | Equipment：LightCycler® 96 |
- AMV Reverse Transcriptase (20 units/μL) was diluted to 0.2 units/μL and then added to the reaction system.
Results
After 0.5 ng of template RNA is added, amplification was confirmed within 15 minutes.
Example of visual determination with the use of LAMP MASTER for Turbidity (Visible Dye)
Q & A
Q：Can DNA be amplified using RNA as template (RT-LAMP)?
A：This can be done by adding AMV Reverse Transcriptase to the reaction system.
What equipment can be used to detect turbidity?
A：The endpoint turbidimeter LT-16 (Product Number: NE4011) can be used.
Q：What equipment can be used for fluorescence detection?
A：A real-time PCR device can be used.
Q：What UV irradiator can be used for visual determination?
A：A UV irradiator with a wavelength range of 240–260 nm or 350–370 nm can be used.
Q：Can DNA amplification be detected by a turbidimeter after addition of 10 x Intercalation Mix?
A：There is no formation of insoluble material (white turbidity) resulting from pyrophosphate formation because 10 x Intercalation Mix contains an intercalator and thermostable pyrophosphatase. As a turbidimeter cannot be used for detection, use a fluorescence detector such as a real-time PCR device.
Q：Can DNA amplification be detected by a turbidimeter after addition of 25 x Visible Dye?
A：Amplification can be detected, but the recommended LAMP reaction time differs for turbidity measurement and visual determination. It is possible to prolong the reaction time, but in these circumstances you should confirm that no nonspecific reaction has occurred, using a negative control.
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For research use or further manufacturing use only. Not for use in diagnostic procedures.
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