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FAQs: LavaLAMP™ DNA Master Mixes

  1. Can the LavaLAMP DNA Master Mix be used for RNA?
    LavaLAMP™ Master Mixes are optimized for DNA targets and have not been optimized with RNA targets. They may work with RNA targets, but we do not have sufficient data to support this application

  2. I’m not sure how to design the primers for my target. What should I do? 
    You need to design a set of 6 primers for each target (FIP, BIP, Loop-F, Loop-B, F3, and B3). We suggest using the free online Eiken PrimerExplorer or available for-purchase, LAMP Designer software, to assist in this process. When developing a new assay, we recommend comparing multiple sets of primers to identify the optimum set.

  3. Can I use 4 primers instead of 6 primers for my target DNA? 
    Yes, 4 primers may be used (FIP, BIP, F3, and B3), the assay, however, will be slower and the sensitivity of the assay will most likely be compromised.

  4. Can I use other dyes besides the Green Fluorescent Dye available in the LavaLAMP™ DNA Master Mix with Dye?
    Yes. Syto-13 (Molecular Probes) and EvaGreen (Biotium) have both been tested with the LavaLAMP DNA Master Mix and they perform well.

  5. Is okay to add the dye to the LavaLAMP Master Mix and store it overnight at 4°C?  
    No. Add the dye to the reaction mix and run the reaction promptly.

  6. Is the 90°C preheating step necessary, and if not when would I use it?
    No. The preheating step may accelerate the reaction when using purified target as input, resulting in lower time to result (TTR) values.

  7. How many copies of template are in the positive control? Do I need to prepare dilutions?
    The control template contains 25,000 copies/µL or 0.10 pg/µL and can be used directly without preparing dilutions.

  8. Can I perform the preheat step at temperatures > 95°C? 
    No. The preheat step should be carried out ≤90°C for up to 5 min.

  9. I’m analyzing complex samples, such as blood, urine, stool, and saliva. Can I use crude sample preparations?
    Yes. However, assay-specific performance can be impacted by sample-related inhibition. So the assay should be developed/tested first with purified target DNA before proceeding to crude samples. The amount of crude sample which the assay will tolerate will vary with sample type, and must be determined empirically.

  10. I left the LavaLAMP Master Mix out on the bench overnight. Is it still good? 
    Yes, the LavaLAMP Master Mixes are stable at room temperature overnight. However, we do not have data to support the stability of the combined reagents (LavaLAMP Master Mix, primers and dye) over time at room temperature.

  11. I would like to take this out in the field. Is it possible to lyophilize the Master Mix? 
    Yes. The LavaLAMP Master Mix supplied is formulated to enable lyophilization. For specific lyophilization parameters consult the lyophilization vendor’s user manual.

  12. What if I don’t have a fluorimeter? How else can I monitor the reaction?
    Amplified products can also be monitored by viewing the reaction on an agarose gel or by measuring the turbidity of the reaction at OD600.

  13. Why didn’t I see evidence of amplification? 
    Poor primer design.  Use online software to design a set of 6 primers. Not all primer designs will function well, so test multiple primer sets to achieve best results.

    PCR and Bst DNA Polymerase reaction conditions may have been used.  These reaction conditions are not compatible with the LavaLAMP™ DNA Master Mix.

    The Primer concentrations and ratios were incorrect.
    1. The LAMP Primer Mix, 10X should be:
      1. 16 µM for each FIP and BIP primer
      2. 8 µM for each Loop-F and Loop-B primer
      3. 2 µM for each F3 and B3 primer

    Samples were too dirty or complex. Contaminants in the sample may have inhibited the reaction. Purify the DNA or dilute the sample.

    The incorrect fluorescence settings were selected on the instrument.  Use the “FAM” channel on fluorimeter (See user manual for tested instruments).

  14. Why does the signal appear early in my no template control (NTC)? Contamination?
    Sample or kit reagents may have been contaminated with target. Prepare reactions in separate areas away from samples to minimize cross-contamination.

    Incubation temperature was not optimal. Best results are obtained in the range of 68°C to 74°C, and must be determined for each primer set. The optimal temperature should be chosen based on both positive sample TTR and NTC TTR (ΔTTR).

    Poor primer design. Use online software to design a set of 6 primers.  Test multiple primer sets to identify the best performing primer set.

    Primer concentration may be too high. Do not exceed concentrations recommended in the LavaLAMP Master Mix User Manual (Also see #13 above).

    Reaction was not set up on ice. Non-specific amplification can occur at ambient temperatures.

  15. Why do I see a ladder of bands on my gel and not just one?
    This pattern is normal and represents the various loop concatamers that are generated during the LAMP reaction.

  16. What is an acceptable TTR difference between my sample and NTC (ΔTTR)?
    The ΔTTR is target- and primer set- dependent. Generally, using the lowest target concentration that you can reliably detect, the ΔTTR between the sample and the NTC should be at least 5 minutes.

  17. Why didn’t my reaction work at 74°C, like the positive control? 
    The control reaction has been optimized with the kit-supplied DNA Positive Control LAMP Primer Mix, which functions best at 74°C. Each sample target is unique, requiring properly designed target-specific primers and optimal temperatures for successful amplification. Optimization of reaction temperature with new target and primer sets optimization should be performed to maximize performance.

  18. In my endpoint assay, the NTC signal was the same as my target signal. Why?
    The reaction ran too long and the background NTC signal was allowed to catch up to the maximum sample signal, mostly likely due to primer dimerization or nonspecific primer annealing and extension events.
    See FAQ number 14: Why does the signal appear early in my no template control (NTC)?

  19. Can I run a real-time assay instead of an end-point assay?
    Yes.  A real-time assay is preferred because the reaction progression is easily monitored over time for both the target and NTC samples.

  20. Which instruments can be used to monitor the real-time fluorescent assays?
    See the User Manual for a list of instruments that have been tested at Lucigen. Basically, any instrument capable of maintaining stable reaction temperatures and detecting fluorescence in the FAM (fluorescein) channel will work.

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