The sgRNA cloning vector was obtained from Addgene (Plasmid #41824). sgRNA sequences were selected using CRISPRdirect (http://crispr.dbcls.jp )20 (link). The selected sequences were sgRNA1 (5′-GAGACCATCTACTCTTGCCTTGG -3′), sgRNA2 (5′-AGATAATTACCATGCAACTTGG -3′), and sgRNA3 (5′-GGGTCACACAGAAGACGCCAGG -3′) where proto-spacer adjacent motif (PAM) sequences are underlined. sgRNAs were prepared as previously described6 (link). sgRNA sequences except the first nucleotide and PAM were cloned into sgRNA cloning vectors by inverse PCR using primer pairs sgRNA1F/sgRNA1R for sgRNA1, sgRNA2F/sgRNA2R for sgRNA2, and sgRNA3F/sgRNA3R for sgRNA3 (Supplementary Table 1 ). sgRNA fragments were amplified using T7-added primers (T7sgRNA1F for sgRNA1, T7sgRNA2F for sgRNA2, and T7sgRNA3F for sgRNA3) and sgRNA-R (Supplementary Table 1 ). sgRNAs were synthesized by means of an in vitro transcription reaction using the mMESSAGE mMACHINE T7 Transcription Kit (Ambion). Transcribed RNAs were purified using the MEGAclear RNA Kit (Ambion).
Megaclear rna kit
Manufactured by Thermo Fisher Scientific
The MEGAclear RNA Kit is a product designed for purifying RNA from a variety of sample types. It utilizes a silica-based membrane technology to efficiently capture and elute RNA, providing a reliable and consistent method for RNA isolation and purification.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using megaclear rna kit
1
CRISPR sgRNA Cloning and Synthesis
2
Cloning and Transcription of CRISPR sgRNAs
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Human codon-optimized Cas9 (hCas9) and sgRNA cloning vector were gifted from George Church (Addgene plasmid #41815 and #41824, respectively) [3 (link)]. Specific sgRNA sequences were designed using the CRISPR Design Tool (http://genome-engineering.org/). sgRNAs for
Irx3, Irx5, and Cas9 mRNA were prepared as previously described [20 (link), 21 (link)]. Three sgRNA target sequences were cloned into sgRNA cloning vectors by inverse PCR using primers pairs
Irx3_sg-F/Irx3_sg-R, Irx5_sg1-F/Irx5_sg1-R, and Irx5_sg2-F/Irx5_sg2-R (Table 1 ). In vitrotranscription was performed using the mMESSAGE mMACHINE T7 Transcription Kit (Ambion, Austin, TX). PCR products amplified using T7-added primers (T7-Irx3_sg for
Irx3_sg, T7-Irx5_sg1 for Irx5_sg1, T7-Irx5_sg2 for Irx5_sg2, and Cas9-F and Cas9-R for Cas9 mRNA;
Table 3 Primer sequences ![]()
Irx3, Irx5, and Cas9 mRNA were prepared as previously described [20 (link), 21 (link)]. Three sgRNA target sequences were cloned into sgRNA cloning vectors by inverse PCR using primers pairs
Irx3_sg-F/Irx3_sg-R, Irx5_sg1-F/Irx5_sg1-R, and Irx5_sg2-F/Irx5_sg2-R (
Irx3_sg, T7-Irx5_sg1 for Irx5_sg1, T7-Irx5_sg2 for Irx5_sg2, and Cas9-F and Cas9-R for Cas9 mRNA;
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