The PCR products were transcribed for 6 h at 37 °C using MegaScript kit (Ambion, Kaufungen, Germany), digested for 5 min at 37 °C with TURBO DNAse (Ambion) and purified using MegaClear kit (Ambion). The purified transcription products were precipitated for 16 to 18 h at −20 °C in 100% EtOH, 10% (v/v) 3.0 M sodium acetate (pH 5.3), and resuspended in RNAse-free water [20 (link)]. RNA concentration and purity were assessed using NanoDrop OneC and the profiles were observed after denaturing gel electrophoresis using a 10% Mini-PROTEAN® TBE-Urea Precast gel (Bio-Rad, Hercules, CA, USA) or using the Experion automated electrophoresis system with the RNA StdSens analysis kit (Bio-Rad) after a denaturation at 90 °C for 2 min followed by 5 min in ice.
Mini protean tbe urea precast gel
Manufactured by Bio-Rad
Sourced in United States
The Mini-PROTEAN® TBE-Urea Precast gel is a pre-cast polyacrylamide gel designed for electrophoresis. It is formulated with Tris-Borate-EDTA (TBE) buffer and contains urea. The gel is intended for the separation and analysis of nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Megaclear kit, by Thermo Fisher Scientific (1 mentions)
Megascript kit, by Thermo Fisher Scientific (1 mentions)
Rna stdsens analysis kit, by Bio-Rad (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
Nanodrop onec, by Bio-Rad (1 mentions)
Hiscribe t7 quick high yield rna synthesis kit, by New England Biolabs (1 mentions)
Rnaclean xp kit, by Beckman Coulter (1 mentions)
Typhoon gel scanner, by GE Healthcare (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Wizard sv gel and pcr clean up system, by Promega (1 mentions)
Ribomax large scale rna production systems kit, by Promega (1 mentions)
4 protocols using mini protean tbe urea precast gel
1
RNA Purification and Characterization
2
Optimized crRNA Synthesis for CRISPR
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All CRISPR-RNAs (crRNAs) used in this study are listed in Supplementary Table 6 . To produce the crRNAs, we followed a previously published protocol40 (link). In short, the templates for the crRNAs were ordered as DNA oligonucleotides from Sigma-Aldrich with an appended T7 promoter sequence (listed in Supplementary Table 6 ). These oligos were annealed with a T7-3G oligonucleotide and input in an in vitro transcription (IVT) reaction (HiScribe T7 Quick High Yield RNA Synthesis Kit, NEB, #E2050S). The crRNAs were then purified using Agencourt RNAClean XP Kit (Beckman Coulter, #A63987). The correct size of the crRNAs was confirmed on a Mini-PROTEAN TBE-Urea precast gel (Bio-Rad, #4566033) and the concentration evaluated by NanoDrop. Aliquots of each crRNA at the working concentration were produced to avoid repeated freeze and thaw cycles and stored at −80 °C.
3
Purification and Characterization of CpG-ODN
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CpG-ODN containing compounds were
purified using Mini-PROTEAN TBE-Urea Precast Gels (BIO-RAD) and Mini-PROTEAN
Tetra Cell system. Compounds were loaded into gels in TBE urea buffer
(7:20 compound:loading buffer). Gels were run in TBE buffer at 100
V for 1 h. The resulting gels were imaged using a GE Typhoon gel scanner.
The desired band was excised, crushed, and eluted into HPLC grade
water overnight at 37 °C. The resulting solution was concentrated
using 3k Amicon Centrifugal Filter Units (EMD Millipore) and filtered
using 0.2 μM cellulose acetate syringe filter (Restek). The
resulting product was desalted using ZipTipC18, analyzed
by MALDI-TOF using 3-hydroxypicolinic acid matrix, and quantified
using a NanoDrop spectrophotometer.
purified using Mini-PROTEAN TBE-Urea Precast Gels (BIO-RAD) and Mini-PROTEAN
Tetra Cell system. Compounds were loaded into gels in TBE urea buffer
(7:20 compound:loading buffer). Gels were run in TBE buffer at 100
V for 1 h. The resulting gels were imaged using a GE Typhoon gel scanner.
The desired band was excised, crushed, and eluted into HPLC grade
water overnight at 37 °C. The resulting solution was concentrated
using 3k Amicon Centrifugal Filter Units (EMD Millipore) and filtered
using 0.2 μM cellulose acetate syringe filter (Restek). The
resulting product was desalted using ZipTipC18, analyzed
by MALDI-TOF using 3-hydroxypicolinic acid matrix, and quantified
using a NanoDrop spectrophotometer.
4
CRISPR sgRNA Template Generation
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To make the single guide RNA (sgRNA), we first PCR amplified a dsDNA template, which contained the consensus sequence from a DNA plasmid (pgRNA-bacteria plasmid from Addgene), using a sgRNA FWD primer that contained a T7 promoter, and sgRNA REV primer. The following thermal cycling conditions were used to generate the PCR template: 98 °C for 3 minutes; 98 °C for 10 seconds; 65 °C for 20 seconds; 72 °C for 15 seconds; go to 98 °C for 10 seconds; 65 °C for 20 seconds; 72 °C for 15 seconds for 29 cycles and 72 °C for 8 minutes. The PCR template was verified using gel electrophoresis (1.5% agarose, 1× TBE buffer, 120 V for 90 minutes) and subsequently purified using the WizardSV Gel and PCR Clean-Up System (Promega) according to the manufacturer's instructions. sgRNA was then transcribed from the PCR template using the RiboMax™ Large Scale RNA Production Systems kit (Promega) according to the manufacturer's instructions. Following transcription, RNA products were purified using the RNeasy MinElute Cleanup Kit (Qiagen) according to the manufacturer's instructions. RNA quality was verified using gel electrophoresis (Mini-Protean TBE-Urea Precast Gels (Bio-Rad), 200 V for 30 minutes). Gels were visualized under UV light in a Biorad ChemiDOCT MP imaging system.
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