Typical RT-LAMP Protocol (NEB #M0439)
Incubate the following reaction at 65°C for 30–60 minutes.
|
Component |
Volume |
Final Concentration |
|---|---|---|
|
10X Isothermal Amplification Buffer (Lyo-compatible) |
2.5 µl |
1X |
|
dNTP Mix (10 mM) |
3.5 µl |
1.4 mM |
|
FIP/BIP Primers (25X) |
1 µl |
1.6 µM |
|
F3/B3 Primers (25X) |
1 µl |
0.2 µM |
|
LoopF/B Primers (25X) |
1 µl |
0.4 µM |
|
Bst 2.0 WarmStart DNA Polymerase (Glycerol-free) (8,000 U/ml) |
1 µl |
320 U/ml |
|
WarmStart RTx Reverse Transcriptase (Glycerol-free) (15,000 U/ml) |
0.5 µl |
300 U/ml |
|
Template |
Variable |
> 10 copies |
|
Nuclease-free Water |
to 25 µl |
|
|
Total Reaction Volume |
25 µl |
General Guidelines:
- A LAMP Primer Mix can be prepared with all 4 or 6 (with Loop) primers. A 25X Primer Mix should contain 40 µM FIP, 40 µM BIP, 5 µM F3, 5 µM B3, 10 µM LoopF, 10 µM LoopB in TE or water.
- Dilute the Bst 2.0 WarmStart DNA Polymerase (Glycerol-free) (NEB #M0402) to 8,000 U/ml and WarmStart RTx Reverse Transcriptase (Glycerol-free) (NEB #M0439) to 15,000 U/ml in 1X Isothermal Amplification Buffer (Lyo-compatible).
- If analyzing via agarose gel electrophoresis or other method requiring opening LAMP reaction vessels, set up a secondary analysis area and equipment to avoid contamination.
- Running a no-template control (NTC) is strongly recommended to ensure amplification specificity.
- If optimization is desired, try titrating Bst 2.0 WarmStart DNA Polymerase (Glycerol-free) (0.04-0.32U/ul), WarmStart RTx Reverse Transcriptase (Glycerol-free), or changing the reaction temperature (50–68°C).