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| - | < | + | <h6>Table 1 PCR program for elongation of knockout cassette for knocking out Far1 and Gpa1 and primers used for elongation.</h6> |
| - | <table border="0" width=" | + | <table border="0" width="500" cols="4"> |
<tr> | <tr> | ||
<td><h3>Repeats</h3> | <td><h3>Repeats</h3> | ||
<td><h3>Temperature</h3> | <td><h3>Temperature</h3> | ||
<td></td> | <td></td> | ||
| - | <td>Duration</td> | + | <td><h3>Duration</h3></td> |
</tr> | </tr> | ||
<td rowspan="3">5x</td> | <td rowspan="3">5x</td> | ||
Revision as of 10:26, 12 August 2012
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 |
