"
Team:TU-Delft/results
From 2012.igem.org
(Difference between revisions)
| Line 1: | Line 1: | ||
| - | {{:Team:TUDelft/Scripts | + | {{:Team:TUDelft/Scripts}}{{:Team:TUDelft/menu}} |
<html> | <html> | ||
<head><title></title> | <head><title></title> | ||
| Line 14: | Line 14: | ||
<br/> | <br/> | ||
<h6>Table 1 PCR program for elongation of knockout cassette for knocking out Far1 and Gpa1 and primers used for elongation.</h6> | <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" style="width: | + | <table border="0" style="width:400px;" cols="4" cellspacing="5"> |
<tr> | <tr> | ||
<td><h3>Repeats</h3> | <td><h3>Repeats</h3> | ||
| Line 52: | Line 52: | ||
<td>2:00</td> | <td>2:00</td> | ||
</tr> | </tr> | ||
| - | < | + | </table > |
| + | <br/> | ||
| + | <table border="0" style="width:300px; font-weight:6px"> | ||
| + | <tr> | ||
<td><b>GPA ko fw</b> TTAGCATCACATCAATAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC</td> | <td><b>GPA ko fw</b> TTAGCATCACATCAATAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC</td> | ||
</tr> | </tr> | ||
| - | <tr | + | <tr > |
<td><b>GPA ko rv</b> TGCATCTTCGGAAACAGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG</td> | <td><b>GPA ko rv</b> TGCATCTTCGGAAACAGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG</td> | ||
| - | |||
| - | |||
| - | |||
</tr> | </tr> | ||
| - | <tr | + | <tr > |
<td><b>FAR1 ko fw</b> ACACAAAGTCTATAGATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTAATGCAGCTGAAGCTTCGTACGC</td> | <td><b>FAR1 ko fw</b> ACACAAAGTCTATAGATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTAATGCAGCTGAAGCTTCGTACGC</td> | ||
</tr> | </tr> | ||
| - | <tr | + | <tr > |
<td><b>FAR1 ko rv</b> GAAAAAAAAAAAAGGAAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTACTAGCATAGGCCACTAGTGGATCTG</td> | <td><b>FAR1 ko rv</b> GAAAAAAAAAAAAGGAAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTACTAGCATAGGCCACTAGTGGATCTG</td> | ||
</tr> | </tr> | ||
| - | <tr | + | <tr > |
<td><b>GPA ko fw short</b> TAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC</td> | <td><b>GPA ko fw short</b> TAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC</td> | ||
</tr> | </tr> | ||
| - | <tr | + | <tr > |
<td><b>GPA ko rv short</b> AGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG</td> | <td><b>GPA ko rv short</b> AGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG</td> | ||
</tr> | </tr> | ||
| - | <tr | + | <tr > |
<td><b>FAR1 ko fw short</b> ATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTAATGCAGCTGAAGCTTCGTACGC</td> | <td><b>FAR1 ko fw short</b> ATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTAATGCAGCTGAAGCTTCGTACGC</td> | ||
</tr> | </tr> | ||
| - | <tr | + | <tr > |
<td><b>FAR1 ko rv short</b> AAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTACTAGCATAGGCCACTAGTGGATCTG</td> | <td><b>FAR1 ko rv short</b> AAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTACTAGCATAGGCCACTAGTGGATCTG</td> | ||
</tr> | </tr> | ||
| - | <tr | + | <tr > |
<td><b>ATF1 ko fw</b> gaaaataaaaaacggCACTTCATCAGTATCACAAATACCATCAATTTATCAGCTCTCATGCAGCTGAAGCTTCGTACGC </td> | <td><b>ATF1 ko fw</b> gaaaataaaaaacggCACTTCATCAGTATCACAAATACCATCAATTTATCAGCTCTCATGCAGCTGAAGCTTCGTACGC </td> | ||
</tr> | </tr> | ||
| - | <tr | + | <tr > |
<td><b>ATF1 ko rv</b> ggttatttacacgacatAATCATATTGTCGAATAATATCAGTCAAGCATCATGTGAGATCTAGCATAGGCCACTAGTGGATCTG </td> | <td><b>ATF1 ko rv</b> ggttatttacacgacatAATCATATTGTCGAATAATATCAGTCAAGCATCATGTGAGATCTAGCATAGGCCACTAGTGGATCTG </td> | ||
</tr> | </tr> | ||
| - | <tr | + | <tr > |
<td><b>ATF1 ko fw short</b> CACTTCATCAGTATCACAAATACCATCAATTTATCAGCTCTCATGCAGCTGAAGCTTCGTACGC </td> | <td><b>ATF1 ko fw short</b> CACTTCATCAGTATCACAAATACCATCAATTTATCAGCTCTCATGCAGCTGAAGCTTCGTACGC </td> | ||
</tr> | </tr> | ||
| - | <tr | + | <tr > |
<td><b>ATF1 ko rv short</b> AATCATATTGTCGAATAATATCAGTCAAGCATCATGTGAGATCTAGCATAGGCCACTAGTGGATCTG </td> | <td><b>ATF1 ko rv short</b> AATCATATTGTCGAATAATATCAGTCAAGCATCATGTGAGATCTAGCATAGGCCACTAGTGGATCTG </td> | ||
</tr> | </tr> | ||
Revision as of 11:04, 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 |
