Author: Long T. Nguyen; Brianna M Smith; Piyush K. Jain
Title: Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection Document date: 2020_4_14
ID: n5kpvsvg_27
Snippet: Cell pellets were collected from the overnight cultures by centrifugation, resuspended in lysis buffer (2M NaCl, 20 mM Tris-HCl, 20 mM imidazole, 0.5 mM TCEP, 0.25 mg/ml lysozyme, and 1mM PMSF, PH = 8), and broken by sonication. The sonicated solution then underwent high speed centrifugation at 40,000 RCF for 45 minutes. The collected supernatant was then run through a Ni-NTA Hispur column (Thermofisher) pre-equilibrated with wash buffer A (2M Na.....
Document: Cell pellets were collected from the overnight cultures by centrifugation, resuspended in lysis buffer (2M NaCl, 20 mM Tris-HCl, 20 mM imidazole, 0.5 mM TCEP, 0.25 mg/ml lysozyme, and 1mM PMSF, PH = 8), and broken by sonication. The sonicated solution then underwent high speed centrifugation at 40,000 RCF for 45 minutes. The collected supernatant was then run through a Ni-NTA Hispur column (Thermofisher) pre-equilibrated with wash buffer A (2M NaCl, 20 mM Tris-HCl, 20 mM imidazole, 0.5 mM TCEP, PH = 8). The column was then eluted with buffer B (2M NaCl, 20 mM Tris-HCl, 300 mM imidazole, 0.5 mM TCEP, PH = 8). The eluted fractions were then pooled together and underwent TEV cleavage overnight at 4 o C (TEV protease was purified using the plasmid pRK793, #8827 from Addgene, a gift from David Waugh Lab).
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