| mutagenic oligos | |||||||||
| FCT ki | |||||||||
| step | primer name | mutations | strand | sequence ( 5,--->3' ) | length | method of mut. | notes | ||
| 1 | 1000? | Y1222F | + | AAGCACCTTACCCCGGGTTCG | 21 | traditional | primes 5' of EcoRI ~1kb before stop codon | ||
| 1 | 1004 | Y1222F | - | CGGAACGCGTCGACGGCCGCTATTGACGATCCTCTGGCTGCTTCTGGAAGCTGATGCTGGCAAAGTTGCTTAAATCC | 77 | traditional | introduce 1222F and restriction sites after TAG stop | ||
| 2 | 1002 | Y1172F | + | GGGTTTGGAGAAGAGTCTTAACTTCATAGATCTGGATTTGGCCAAGG | 47 | quickchange | introduce 1172F and BglI | ||
| 2 | 1003 | Y1172F | - | CCTTGGCCAAATCCAGATCTATGAAGTTAAGACTCTTCTCCAAACCC | 47 | quickchange | introduce 1172F and BglI | ||
| 3 | 1008 | none | - | GGAACGCGTCGACGGCCGGCGGGTACCCTATTGACGATCCTCTGGCTGC | 49 | NA | change sequence after TAG stop for cloning (match MD/JK) | ||
| S307A ki | |||||||||
| step | primer name | mutations | strand | sequence ( 5,--->3' ) | length | method of mut. | notes | ||
| 1 | Irs1 BamHI for | BamHI | + | cctgccagtatggtgggtgggaaaccaggatccttccgg | 39 | quickchange | introduce BamHI | ||
| 1 | Irs1 BamHI back | BamHI | - | ccggaaggatcctggtttcccacccaccatactggcagg | 39 | quickchange | introduce BamHI | ||
| 2 | irs1 s307a 1 | S307A | + | GCATCACTGCCACCGCCCCTGCCAGTA | 27 | quickchange | introduce 307A | ||
| 2 | irs1 s307a 2 | S307A | - | TACTGGCAGGGGCGGTGGCAGTGATGC | 27 | quickchange | introduce 307A | ||
| JBM ki | |||||||||
| step | primer name | mutations | strand | sequence ( 5,--->3' ) | length | method of mut. | notes | ||
| 1 | IRS1 JnkBMNHe | L856/858G+NheI | + | cccacaagggggtccgggggggatcccaaggctagcacc | 39 | quickchange | introduce 856/858G and NheI site; leaves BamHI intact | ||
| 1 | IRS1 JnkBMNHe Comp | L856/858G+NheI | - | ggtgctagccttgggatcccccccggacccccttgtggg | 39 | quickchange | introduce 856/858G and NheI site; leaves BamHI intact | ||