- I am using the ingredients of 20mM Tris HCl, 10mM (NH4)2SO4, 50 mM KCl, 8mM MgSO4, 0,1 % Tween, 0.8 M Betaine, 1.4 mM dNTPs mix, FIP/BIP primers 1.6 µM, F3/B3 0.2 µM, Loop F/Loop B 0.4 µM, Bst polymerase 8 U, dye(50X) 0.5 µl. Everything was properly mixed and kept in thermal cycler with set temperature of 65 0 C for nearly 100 cycle. I could not able to see the amplification phase, most of the time it follows flat curve and some times with noisy. I am unsure on the reaction part, really it happened or not.
- The primers works perfectly with the commercial kit, when I implement with real mix of ingredients is the problematic.
- So please share your knowledge to solve this problem in LAMP analysis on real time detection. any suggestions on components change, concentration change, thermal cycle change?
Hi,
- Could you provide some background on how you designed your LAMP primers? I always recommend using a software design tool (I personally love PrimerExplorer). Using a software makes primer design so much easier as it helps to suggest primers that will potentially work, mitigating those outside the accepted parameters so you don't have to. I would say it is easier and smarter to let the software do the brunt of the hard work when it comes to the design, and you then validating the primer. (Saves you the headache as well).
- Your primer used, does it amplify bands using conventional PCR+ gel electrophoresis? Is your target area amplified? Is it possible that other non-target species is being amplified (thus the noise).
- There is also a possibility that during the experimentation, cross contamination occurred. LAMP is highly sensitive to cross contamination.You might have carried over DNA from your previous assay onto this one. Ensure that your DNA extraction workspace differs from where you carry out the LAMP assay and make sure your workspace is always sterilized, and you should make sure to open the reaction tubes at a different place compared to the incubation area to prevent aerosol contamination.
- What is your target species? Is there previous literature that you can adapt for your experiment conditions? It would be easier just to adapt a published literature (cite them of course).
- You can try to optimise the concentration levels (trial and error) and try reducing the temperature and primer concentration. See if that helps.
- Another way is to start with a fresh batch of chemicals and see if it gives results.
Goodluck Nivedhitha Narayanan
Hi ,
Thank you Lavanya Malini
I used the LAMP designer software, for designing primer set, and found it works perfectly with commercial kit mix, starts exponential phase at 20/80 cycles with high delta Rn value. When I try to make own reagent mix with optimized concentration of individual chemicals, it gone completely failure. I had changed the slight concentration of primers, dNTPs, Bst enzyme in different trials,still not working. Eventhough, it shoes DNA band at gel electrophoresis, I am doubtful it could be of unreacted DNA which prominently visible. I am trying my best in different ways.
Thank you for the suggestions..
Regards
Nivedhitha
Hi. Good news is your primers work. Is there a reason why you prefer to use your own mix? Maybe try a new fresh batch of chemicals. Start again with newly diluted chemicals and see if there's a reaction. Maybe even try a diff primer ratio. I used 8:1:4 (Fip:F:LF).
I hope you find a solution.
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