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Team:TU-Delft/results
From 2012.igem.org
Making knockout strain (far1::kanmx, gpa1::URA) and preparation of knocking out atf1
Week of 05-07-12
For making a functional knockout, yeast strains BY4741; Mat a; his3D1; leu2D0; met15D0; ura3D0; YJL157c::kanMX4 and BY4741; Mat a; his3D1; leu2D0; met15D0; ura3D0; YHR005c::kanMX4 were used (Euroscarf). Also knockout cassette pUG72 is used (Euroscarf). The LoxP-Ura-LoxP is elongated using the pFx polymerase protocol. The PCR program and primer sequences in table 1 yielded a product which is put on a gel shown in figure 1. Here a band can be recognized between 2000 and 1500 nucleotides, which corresponds to the 1669 nucleotide PCR product.
Table 1 PCR program for elongation of knockout cassette for knocking out Far1 and Gpa1 and primers used for elongation.
Repeats | Temperature | Duration |
5x | 95 | Melting | 2:00 |
| 51 | Annealing | 1:00 | |
| 68 | Elongation | 2:00 | |
| 25x | 95 | Melting | 2:00 |
| 61 | Annealing | 1:00 | |
| 68 | Elongation | 2:00 |
| GPA ko fw TTAGCATCACATCAATAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC |
| GPA ko rv TGCATCTTCGGAAACAGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG |
| FAR1 ko fw ACACAAAGTCTATAGATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTAATGCAGCTGAAGCTTCGTACGC |
| FAR1 ko rv GAAAAAAAAAAAAGGAAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTACTAGCATAGGCCACTAGTGGATCTG |
| GPA ko fw short TAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC |
| GPA ko rv short AGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG |
| FAR1 ko fw short ATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTAATGCAGCTGAAGCTTCGTACGC |
| FAR1 ko rv short AAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTACTAGCATAGGCCACTAGTGGATCTG |
| ATF1 ko fw gaaaataaaaaacggCACTTCATCAGTATCACAAATACCATCAATTTATCAGCTCTCATGCAGCTGAAGCTTCGTACGC |
| ATF1 ko rv ggttatttacacgacatAATCATATTGTCGAATAATATCAGTCAAGCATCATGTGAGATCTAGCATAGGCCACTAGTGGATCTG |
| ATF1 ko fw short CACTTCATCAGTATCACAAATACCATCAATTTATCAGCTCTCATGCAGCTGAAGCTTCGTACGC |
| ATF1 ko rv short AATCATATTGTCGAATAATATCAGTCAAGCATCATGTGAGATCTAGCATAGGCCACTAGTGGATCTG |
