Lyophilization of RT-LAMP reagents

A mixture of Bst 2.0 WarmStart® DNA Polymerase (16 U), WarmStart® RTx Reverse Transcriptase (15 U), RNaseOUT™ (80 U) and Antarctic thermolabile UDG (2 U) in dialysis buffer (200 μL, 10 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA10 and 0.1% Triton X-100) was placed in an ultrafiltration membrane (10 kDA cut-off limit, Millipore, Billerica, MA). Samples were centrifuged at 14,000 x g for ca. 15 min to concentrate (down to ~5 μL) and to remove glycerol. 10X LAMP primers (5 μL), dNTPs (10 mM each, 7 μL) and glycerol free enzyme mix (5 μL) were combined and lyophilized and supplemented with 1.1X-LAMP rehydration buffer (22 mM Tris-HCl, pH 8.8, 55 mM KCl, 11 mM (NH₄)₂SO₄, 8.8 mM MgSO₄, 0.11% Tween® 20, 1.1 mM DTT). Plasma samples (5 μL) were mixed with 1.1X rehydration buffer (45 μL) and incubated at 65 °C for 45 min. The resulting fluorescence signal was observed by blue LED excitation (470 nm) through an orange filter.