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Alice:Whole Genome Bisulfite Sequencing Lab Notes/methylation reporter
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==cloning protocol== Sox2se-CPG-TV plasmid maxi-prep: purchased these two plasmids from addgene: SOX2se-CPG-TV (Plasmid #70155); GAPDH-CPG-TV (Plasmid #70148) shake the plasmids in 3ml AmpR LB for 12 hours at 37C, then transfer to 300ml AmpR LB media and shake overnight at 37C use Qiagen maxi-prep kit to extract plasmid DNA from the overnight culture take 100ug of the extracted DNA of Sox2SE sample, and perform overnight digestion with restriction enzyme BamHI donor plasmid: Sox2SE Insert sox2 gRNA into cas9-mcherry plasmid: digest p2U6-pCAG-Cas9-mChery plasmid with BaeI ligate Sox2SE oligo with T4 ligase transform into TOP10 or Sblt3 chemical competent cells plate overnight pick single clone and shake in 3ml AmpR LB media overnight Sanger sequencing to double check for correct insert Notes: To use with p2U6-pCAG-Cas9-mcherry plasmid (cas9 and gRNA on one plasmid construct): Forward: N1-N20-GTTTT Reverse: N20-N1-CGGTG Published sequences: D1 SOX2-SE Crispr CACCGCCAGCTTTCCGAGCCAGATG D2 SOX2-SE Crispr AAACCATCTGGCTCGGAAAGCTGGC Oligos ordered: SOX2-SE-F: CCAGCTTTCCGAGCCAGATGGTTTT SOX2-SE-R: CATCTGGCTCGGAAAGCTGGCCGGTG CRISPR and gRNA plasmid: cas9+sox2se
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