here we try this RT-LAMPseq protocol on real saliva sample (4 plex with 6 positives and 4 négatives on each). for the first experiment, nothing usable in fastq.
We Don't know if the LAMP rxn work or if it's because of later PCR.
We use B set of primers and follow the protocol…
We prepare 200µL RT-LAMP rxn and add 300µL of Saliva (500µL final with paper component concentration) --> 65 °C for 30 minutes to react, and to 95°C for 10 minutes to sterilize
1- we do not see the expected smear at the end of LAMP. can we view it on a bioAnalyzer device? if not, should you make an agarose gel (what conditions and how many inputs on the gel)?
2- should we purify the LAMP before the 1st PCR? what are the PCR cycling conditions (time and temperature) for the first and second PCR?
3- how we can control the first and second PCR? what should we observe on a gel ?
does anyone have answers to these questions or can help us?