User:Hattie Chung/Notebook/Intron Expression of mStaple/2010/01/11

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Entry title Intron will be inserted between positions 2813 and 2814 on the original mKate plasmid (not renumbered) PCR Primers Exon1fwd: 5’-AAGCTAACCGGTCGCCACCATGGTGTCTAAG-3’ Length 31; GC 57.9%; Tm 55.8C (65.9C, entire oligo) Extra sequences AgeI Site Kozak sequence and mKate 2537 – 2562 Exon1rev1: 5’-GCACCCGCCATCCAGCCTGTCCTTTGGACCACTTACCTCAGGGAAGGACTGCTTAAAG -3’ Length: 58; GC 50.0%; Tm 55.1C (64.5C, entire oligo) First half of mStaple Reverse-complement of GTAAGT (Donor Site) Reverse Complement of mKate 2792-2813 Exon1rev2: 5’-CTAATGCTCGAGAGTTAGTACCACGCCCACGCCGATGCACCCGCCATCCAGCC-3’ Length: 53; GC 76.5%; Tm 62.9C Extra Sequences XhoI site Branch Point, Reverse Complement Second half of mStaple Reverse complement of portion of First half of mStaple Exon2fwd: 5’-CAGGAGCTCGAGTCTTCTTTTTTTTTTTCACAGGGCTTCACATGGGAGAGAG-3’ Length 52; GC 57.9%; Tm 55.1C (66.4C, entire oligo) Extra Sequences XhoI site Poly-Pyrimidine Tract Acceptor Site mKate 2814-2832 Exon2rev1: 5’-GTACTTGCGGCCGCTCAATTAAGTTTGTGCCCCAG-3’ Extra Sequences NotI site Reverse Complement of mKate 3236 – 3257 Length 35; GC 40.0%; Tm 56.4C (67.4C, entire oligo) mKate no mStaple negative control: PCR with Exon1fwd + Exon2rev Gata3 mStaple (Length 66 bp): 5’- AAGCTTGGTCCAAAGGACAGGCTGGATGGCGGGTGCATCGGCGTGGGCGTGGTACTAACTCTCGAG-3’ HindIII site, Gata3 mStaple, Branch Point, XhoI site Reverse Complement: 5’-CTCGAGAGTTAGTACCACGCCCACGCCGATGCACCCGCCATCCAGCCTGTCCTTTGGACCAAGCTT-3’