876 pJet1.2-Dr-Muts
PCR 3 fragments from genomic DNA
Fragment 1
874-Nde1-F2
CAGCGGCCTGGTGCCGCGCGGCAGC CATATGCGAGCAGTTTCCCCCACCAAAG
874-R1
TCGCCGAGCGTTTCCCGAATCTCAC
(GTGAGATTCGGGAAACGCTCGGCGA)
Fragment 2:
874-seq-F1: TGA GAT TCG GGA AAC GCT C
847-R2
GGGCGCGGTCTTTGAGGGTCGCAATC
(GATTGCGACCCTCAAAGACCGCGCCC)
Fragment 3:
874-seq-F2: AGA TTG CGA CCC TCA AAG AC
874-Sal1-R2
(CTGGGGGAGGGCCATGAATCCCGG GTCGACAAGCTTGCGGCCGCACTCGAGCACC)
GGTGCTCGAGTGCGGCCGCAAGCTTGTCGACCCGGGATTCATGGCCCTCCCCCAG
1st PCR
2nd PCR
mix 3 fragments, and gel purified, PCR using
874-Nde1-F2
CAGCGGCCTGGTGCCGCGCGGCAGC CATATGCGAGCAGTTTCCCCCACCAAAG
874-Sal1-R2
(CTGGGGGAGGGCCATGAATCCCGG GTCGACAAGCTTGCGGCCGCACTCGAGCACC)
GGTGCTCGAGTGCGGCCGCAAGCTTGTCGACCCGGGATTCATGGCCCTCCCCCAG
PCR screening using
874-seq-F1: TGA GAT TCG GGA AAC GCT C
847-R2
GGGCGCGGTCTTTGAGGGTCGCAATC
(GATTGCGACCCTCAAAGACCGCGCCC)
miniprep digest with Nde1+ Xho1
