I have tried the Tomita et al concentrations of Calcein and MnCl2, the Goto et al concentrations in many attempts to try and prepare home made Eiken Detection Reagent (because i ran out and it takes forever to be shipped). Is there anyone who know exactly what goes in the Eiken Detection Reagent?
The pH of home made attempts can range from 6-8 (in order not to fluoresce) but still i find there must be some buffering agent missing because i get very spontaneous and sudden signal from samples where I wouldnt expect to see signal (together with PC).. Also I get irreproducibe results and observe an overall stochasticity.
So far I have tried dissolving Calcein in DMSO and MnCl in H2O or Tris-HCl pH7.5-8 and then supplement with H2O or DMSO or different combinations of both (1/4 H2O 3/4 DMSO, half half etc..)
I am not happy with anything and believe I m missing something Eiken Detection Reagent has in it...
Originally Posted by deen23
I'm trying to make a fluorescent detection reagent using calcein and MnCl2 (based on the Eiken LAMP Kit). It was hard to find out from papers how people have made their own, but I found two pieces of information:
1. The final concentration of calcein in a 25uL rxn mix should be 25uM, and of MnCl2 should be 0.5mM.
2. Calcein is dissolved in DMSO, and then mixed with MnCl2 dissolved in water.
I've tried (with both MnCl2 tetrahydrate and anhydrous), and unfortunately the final solution starts fluorescing immediately. I was wondering if anyone was successful in making this?