The homogenization buffer was prepared by adding 2.5 mL of 2 M KCl (RNase free; Fisher Scientific, #AM9640G), 2.5 mL of 1 M Tris (pH 7.4; ThermoFisher Scientific, #17,926), 500 µl of NP-40 (ThermoFisher Scientific, #28,324) and 600 µl of 1 M MgCl2 (RNase free; Fisher Scientific, #AM9530G) into a 50 mL Falcon tube. The volume was adjusted to 50 mL with DNase/RNase free water and vortexed until all contents were completely dissolved. The supplemented homogenization buffer (HB-S) was always prepared fresh prior to homogenization by adding 50 µl of protease inhibitor (VWR, #97,063–970), 5 µl of 1 M DTT (VWR, #97,061–340), 25 µl of RNasin (VWR, #PAN2615), 50 µl of Heparin (100 mg/ml; Fisher Scientific, #BP252420) and 100 µl of cycloheximide (5 mg/ml; Fisher Scientific, #AC357420050) in a 15 ml Falcon tube. Finally, the volume was adjusted to 5 mL with HB buffer.
Am9640g
Manufactured by Thermo Fisher Scientific
The AM9640G is a high-performance analytical instrument designed for laboratory applications. It is capable of performing advanced analytical techniques, but a detailed description of its core function is not available without the risk of extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Am9530g, by Thermo Fisher Scientific (3 mentions)
Np 40, by Thermo Fisher Scientific (1 mentions)
Ac357420050, by Avantor (1 mentions)
Purexpress in vitro protein synthesis kit, by New England Biolabs (1 mentions)
Nuclease free water, by Thermo Fisher Scientific (1 mentions)
Protector rnase inhibitor, by Roche (1 mentions)
4 protocols using am9640g
1
Preparation of Supplemented Homogenization Buffer
2
Preparation of High Salt Buffer
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To prepare the high salt buffer, 7.5 mL of 2 M KCl (RNase free; Fisher Scientific, #AM9640G), 2.5 mL of 1 M Tris (pH 7.4; ThermoFisher Scientific, #17,926), 600 µl of 1 M MgCl2 (Rnase free; Fisher Scientific, #AM9530G), and 500 µl of NP-40 (ThermoFisher Scientific, #28,324) were added in a 50 mL Falcon tube. The final volume was made up to 50 mL with DNase/RNase free water and vortexed to completely dissolve all contents. The high salt buffer supplemented (HSB-S) was prepared fresh prior to the washing step by adding together 10 µL of protease inhibitor (VWR, #97,063–970), 40 µl of cycloheximide (5 mg/ml; Fisher Scientific, #AC357420050) and 5 µL of RNasin (VWR, #PAN2615) in a 2 mL Eppendorf tube and the volume was adjusted to 2 mL with HSB.
3
In Vitro Protein Synthesis Protocol
4
NASBA-Based RNA Amplification and Detection
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For target RNA amplification with NASBA, 2.01 μL 3X reaction buffer (Life Sciences, NEC-1-24;), 0.99 μL 6X nucleotide mix (Life Sciences, NEC-1-24), 0.03 μL Protector RNase Inhibitor (Roche), 0.12 μL of each NASBA primer (12.5 mM, IDT), 0.15 μL nuclease-free water (Thermo Fisher, 10977015), and 1.2 μL target RNA were mixed at 4°C and heated to 65°C for 2 min, followed by a 10-minute incubation at 41°C. 1.5 μL Enzyme Mix (Life Sciences, NEC-1-24) was then added to the reaction for a final volume of 6 μL. After mixing, the reaction was incubated at 41°C for 2 hours. For a 35 μL two-pot reaction, 5 μL of the NASBA amplified RNA product was combined with 0.7 μL of RNA aptaswitch and 10X DFHBI-1T dye mix. This reaction was incubated and measured at 37 °C for an additional 2 hr. The 10X DFHBI-1T dye mix consists of 40 μM DFHBI-1T (Lucerna, 410), 40 mM HEPES (Gibco™, 15630080), pH 7.4, 100 mM KCl (Invitrogen™, AM9640G), and 5 mM MgCl2 (Invitrogen™, AM9530G).
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