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. Author manuscript; available in PMC: 2014 Mar 30.
Published in final edited form as: Nat Protoc. 2013 Oct 24;8(11):2281–2308. doi: 10.1038/nprot.2013.143

TABLE 1.

Primer sequences for sgRNA cloning and validation.

Step Primer Sequence (5′-3′) Purpose
5A(iii) U6-Fwd GAGGGCCTATTTCCCATGATTCC Amplify any U6-sgRNA
5A(iii) U6-Rev AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAcNNNNNNNNNNNNNNNNNNNCCGGTGTTTCGTCCTTTCCACAAG Amplify specifically designed U6-sgRNA; N is the reverse complement of target; appended cytosine (complementary to appended guanine) in lowercase
5B(i) sgRNA-top CACCgNNNNNNNNNNNNNNNNNNN Clone sgRNA into pSpCas9(BB); appended guanine in lowercase
5B(i) sgRNA-bottom AAACNNNNNNNNNNNNNNNNNNNc Clone sgRNA into pSpCas9(BB); appended cytosine (complementary to appended guanine) in lowercase
117 pUC-Fwd (M13 -20 primer) GTAAAACGACGGCCAGT Sanger sequencing of modified genomic regions cloned into pUC19
117 pUC-Rev (M13 -26 primer) CAGGAAACAGCTGTAAC Sanger sequencing of modified genomic regions cloned into pUC19