High salt buffer was prepared by adding 2.5 mL of 1 M Tris (pH 7.4; ThermoFisher Scientific, #17926), 7.5 mL of 2 M KCl (RNase free; Fisher Scientific, #AM9640G), 600 μL of 1 M MgCl2 (RNase free; Fisher Scientific, #AM9530G), and 500 μL of NP-40 (ThermoFisher Scientific, #28324) in a 50 mL Falcon tube. The final volume was brought to 50 mL with DNase/RNase free water and vortexed. The high salt buffer supplemented (HSB-S) was prepared before the washing step by adding 40 μL of cycloheximide (5 mg/mL; Fisher Scientific, #AC357420050), 10 μL of protease inhibitor (VWR, #97063-970), and 5 μL of RNasin (VWR, #PAN2615), in a 2 mL Eppendorf tube and the volume made up to 2 mL with HSB. This solution was prepared fresh before the washing step.
Ac357420050
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The AC357420050 is a laboratory equipment product. It serves as a core function for laboratory applications. No further details are available to provide an unbiased and factual description while maintaining conciseness.
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2 protocols using ac357420050
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High Salt Buffer Preparation and Supplementation
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.
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