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Team:TU-Delft/results
From 2012.igem.org
(Difference between revisions)
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<table id="tbtext"> | <table id="tbtext"> | ||
<tr> | <tr> | ||
| - | <td><b>GPA ko fw</b> TTAGCATCACATCAATAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC</td> | + | <td><b>GPA ko fw</b><br/> TTAGCATCACATCAATAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC</td> |
</tr> | </tr> | ||
<tr > | <tr > | ||
| - | <td><b>GPA ko rv</b> TGCATCTTCGGAAACAGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG</td> | + | <td><b>GPA ko rv</b><br/> TGCATCTTCGGAAACAGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG</td> |
</tr> | </tr> | ||
<tr > | <tr > | ||
| - | <td><b>FAR1 ko fw</b> ACACAAAGTCTATAGATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTAATGCAGCTGAAGCTTCGTACGC</td> | + | <td><b>FAR1 ko fw</b><br/> ACACAAAGTCTATAGATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTAATGCAGCTGAAGCTTCGTACGC</td> |
</tr> | </tr> | ||
<tr > | <tr > | ||
| - | <td><b>FAR1 ko rv</b> GAAAAAAAAAAAAGGAAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTACTAGCATAGGCCACTAGTGGATCTG</td> | + | <td><b>FAR1 ko rv</b><br/> GAAAAAAAAAAAAGGAAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTACTAGCATAGGCCACTAGTGGATCTG</td> |
</tr> | </tr> | ||
<tr > | <tr > | ||
| - | <td><b>GPA ko fw short</b> TAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC</td> | + | <td><b>GPA ko fw short</b><br/> TAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC</td> |
</tr> | </tr> | ||
<tr > | <tr > | ||
| - | <td><b>GPA ko rv short</b> AGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG</td> | + | <td><b>GPA ko rv short</b><br/> AGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG</td> |
</tr> | </tr> | ||
<tr > | <tr > | ||
| - | <td><b>FAR1 ko fw short</b> ATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTAATGCAGCTGAAGCTTCGTACGC</td> | + | <td><b>FAR1 ko fw short</b><br/> ATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTAATGCAGCTGAAGCTTCGTACGC</td> |
</tr> | </tr> | ||
<tr > | <tr > | ||
| - | <td><b>FAR1 ko rv short</b> AAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTACTAGCATAGGCCACTAGTGGATCTG</td> | + | <td><b>FAR1 ko rv short</b><br/> AAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTACTAGCATAGGCCACTAGTGGATCTG</td> |
</tr> | </tr> | ||
<tr > | <tr > | ||
| - | <td><b>ATF1 ko fw</b> gaaaataaaaaacggCACTTCATCAGTATCACAAATACCATCAATTTATCAGCTCTCATGCAGCTGAAGCTTCGTACGC </td> | + | <td><b>ATF1 ko fw</b><br/> gaaaataaaaaacggCACTTCATCAGTATCACAAATACCATCAATTTATCAGCTCTCATGCAGCTGAAGCTTCGTACGC </td> |
</tr> | </tr> | ||
<tr > | <tr > | ||
| - | <td><b>ATF1 ko rv</b> ggttatttacacgacatAATCATATTGTCGAATAATATCAGTCAAGCATCATGTGAGATCTAGCATAGGCCACTAGTGGATCTG </td> | + | <td><b>ATF1 ko rv</b><br/> ggttatttacacgacatAATCATATTGTCGAATAATATCAGTCAAGCATCATGTGAGATCTAGCATAGGCCACTAGTGGATCTG </td> |
</tr> | </tr> | ||
<tr > | <tr > | ||
| - | <td><b>ATF1 ko fw short</b> CACTTCATCAGTATCACAAATACCATCAATTTATCAGCTCTCATGCAGCTGAAGCTTCGTACGC </td> | + | <td><b>ATF1 ko fw short</b><br/> CACTTCATCAGTATCACAAATACCATCAATTTATCAGCTCTCATGCAGCTGAAGCTTCGTACGC </td> |
</tr> | </tr> | ||
<tr > | <tr > | ||
| - | <td><b>ATF1 ko rv short</b> AATCATATTGTCGAATAATATCAGTCAAGCATCATGTGAGATCTAGCATAGGCCACTAGTGGATCTG </td> | + | <td><b>ATF1 ko rv short</b><br/> AATCATATTGTCGAATAATATCAGTCAAGCATCATGTGAGATCTAGCATAGGCCACTAGTGGATCTG </td> |
</tr> | </tr> | ||
</table> | </table> | ||
Revision as of 11:38, 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 |
