Team:TU-Delft/results

From 2012.igem.org

(Difference between revisions)
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<td>94</td>
<td>94</td>
<td>Melting</td>
<td>Melting</td>
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<td>1:00</td>
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</tr>
 +
<tr>
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<td>56</td>
 +
<td>Annealing</td>
<td>1:00</td>
<td>1:00</td>
</tr>
</tr>
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</tr>
</tr>
<tr>
<tr>
-
<td>H2O</td><td>25 µl</td>
+
<td>H<sub>2</sub>O</td><td>25 µl</td>
</tr>
</tr>
</table>
</table>
 +
<br/><br/>
 +
<p>The PCR product is put on gel, shown in figure 2. Here a band can be observed in lane 3 with a length of ~4000 nt. This PCR product is inconsistent with both the URA gene (~3000 nt) or the original gene (2000 nt). We ordered new primers, using different regions of the gene. Also primers checking the insert where redesigned.</p>
 +
<br/>
 +
<img src="https://static.igem.org/mediawiki/igem.org/6/64/AgarosegelinTAE_2.png"  align="center" width="250" height="300" />
 +
<h6>Figure 2 0.8% Agarose gel in TAE, 40 minutes 80 V. In the picture can be seen:  1 DNA smartladder, 2 PCR product with DNA of colony 1, 3 PCR product with DNA of colony 2, 4 PCR product with DNA of colony 3.</h6>
 +
<br/>
 +
<br/>
 +
<h3>Week of 20-07-12</h3>
 +
<br/>
 +
<p>To check whether the new primers were working, colony PCR of wildtype yeast was performed. New primers were ordered for the A and D primers, called confident primers (conf). No product occurred in the negative control, PCR of wildtype yeast with the AB and CD primers was also performed. Before initiation the colonies were boiled for 10 minutes in a PCR tube in 2 mM NaOH solution. Conditions according to table 6 were used, using the Phusion polymerase. No results were seen on gel.</p>
 +
<br/>
 +
<h6>Table 6 Phusion PCR reaction.</h6>
 +
<br/>
 +
<table id="tbtext" >
 +
<tr>
 +
<th>Repeats</th>
 +
<th>Temperature</th>
 +
<th></th>
 +
<th>Duration</th>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<td>98</td>
 +
<td>Melting</td>
 +
<td>0:30</td>
 +
</tr>
 +
<tr>
 +
<td rowspan="3">30x</td>
 +
<td>98</td>
 +
<td>Melting</td>
 +
<td>0:10</td>
 +
</tr>
 +
<tr>
 +
<td>57</td>
 +
<td>Annealong</td>
 +
<td>0:30</td>
 +
</tr>
 +
<tr>
 +
<td>72</td>
 +
<td>Elongation</td>
 +
<td>1:15</td>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<td>72</td>
 +
<td>Elongation</td>
 +
<td>10:00</td>
 +
</tr>
 +
</table>
 +
<table id="tbtext" >
 +
<tr>
 +
<td><b>GPA1 A </b>TCTGCGTATTCTTCCTTGTAGAAAT</td>
 +
</tr>
 +
<tr>
 +
<td><b>KO B </b>CGCCAAGGGTAGAGATCCTAAG</td>
 +
</tr>
 +
<tr>
 +
<td><b>KO C </b>CTTCACGCAGGATGACAGTTC</td>
 +
</tr>
 +
<tr>
 +
<td><b>GPA1 D</b>GAATTCGAGATAATACCCTGTCCTT</td>
 +
</tr>
 +
<tr>
 +
<td><b>GPA1 A conf </b>CGTCCTTCTGCGTATTCTTCC</td>
 +
</tr>
 +
<tr>
 +
<td><b>GPA1 D conf </b>CCGAGTATTTACCAGGGAGAAG</td>
 +
</tr>
 +
</table>
 +
 +
<br/><br/>
 +
<p>To check whether the problem was due to the treatment we performed a reaction with 18SrDNA Eukaryotic primers: EukAt & Euk516GC which amplifies a region of 563 nt. First a PCR reaction with taq mastermix was performed and pretreatment of the colonies was dissolving in 10 µl 2mM NaOH and taking 3 µl of this mixture as DNA template. 0.25 µl primers were added. The PCR product was put on gel which is shown in figure 3. PCR reactions were performed on wildtype yeast. A band of ~2000 nt can be observed in lane 3 which corresponds to the length of the gene. Further bands of ~500 nt can be observed in lane 2 and 8 which correspond to the expected Euk516GC primer product. Further can be seen that the band in lane 8 is lighter. This PCR reaction was performed on a 15 minute pretreated wildtype colony. Longer pretreatment will make worse the outcome of the reaction.
 +
</p>
 +
<br/><br/>
 +
<h6>Table 7 Taq PCR reaction on wildtype yeast and knock-out colonies</h6>
 +
<table id="tbtext">
 +
<tr>
 +
<th>Repeats</th>
 +
<th>Temperature</th>
 +
<th></th>
 +
<th>Duration</th>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<td>94</td>
 +
<td>Melting</td>
 +
<td>4:00</td>
 +
</tr>
 +
<tr>
 +
<td rowspan="3">35x</td>
 +
<td>94</td>
 +
<td>Melting</td>
 +
<td>0:30</td>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<td>Annealing </td>
 +
<td>4:00</td>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<td>55</td>
 +
<td>Elongation</td>
 +
<td>0:40</td>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<td>72</td>
 +
<td>Elongation</td>
 +
<td>10:00</td>
 +
</tr>
 +
</table>
 +
<table id="tbtext" >
 +
<tr>
 +
<td><b>GPA1 A (old) </b>TCTGCGTATTCTTCCTTGTAGAAAT</td>
 +
</tr>
 +
<tr>
 +
<td><b>GPA1 D (old) </b>GAATTCGAGATAATACCCTGTCCTT</td>
 +
</tr>
 +
<tr>
 +
<td><b>GPA1 A conf </b>CGTCCTTCTGCGTATTCTTCC</td>
 +
</tr>
 +
<tr>
 +
<td><b>GPA1 D conf </b>CCGAGTATTTACCAGGGAGAAG</td>
 +
</tr>
 +
<tr>
 +
<td><b>KO B </b>CGCCAAGGGTAGAGATCCTAAG</td>
 +
</tr>
 +
<tr>
 +
<td><b>KO C </b>CTTCACGCAGGATGACAGTTC</td>
 +
</tr>
 +
</table>
</div>
</div>
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Revision as of 13:02, 21 August 2012

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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

Figure 1 1% agarose in TAE ~45 run on 80 Volts. In the picture can be seen: 1 SmartLadder, 2 Far1 short PCR product, 3 Far1 long PCR product, 4 Gpa1 short PCR product, 5 Gpa1 long PCR product, 6 Atf1 short PCR product, 7 Atf1 long PCR product.

Gel extraction of the GPA1 PCR product is performed using the Qiagen gel extraction kit. The end concentration of this step is measured to be 167.2 ng/µl using the nanodrop nucleotide program.


The PCR reactions using FAR1 and ATF1 primers with the same conditions as table 1 but with the PCR program according to table 2 is performed yielding no result.



Table 2 PCR program for elongation of knockout cassette for knocking out Far1 and Gpa1 and primers used for elongation.
Repeats Temperature Duration
30 x 95 Melting 2:00
60/62/66/69 Annealing 1:00
68 Elongation 2:00

Drop out medium is made and solutions for transformations in yeast strains is prepared. The formula of the dropout media can be seen in the media protocol.



Week of 09-07-12

Yeast strain BY4741; Mat a; his3D1; leu2D0; met15D0; ura3D0; YJL157c::kanMX4 is transformed with the GPA1 ko PCR product, according to the yeast transformation protocol. 100 ng of DNA is used for transformation. After 3 days 3 colonies could be seen on the plate. There where zero colonies on the control plate.
To check whether the knock-out reaction was successful, colony PCR of the three colonies where performed. Primers A and D bind to 5’ upstream and 3’ downstream regions of the GPA1 gene, the B and C primers anneal in the URA gene which is knocked in. For the first reaction, only AD reaction is tried A negative control is the yeast strain without transformation. Before initiation the colonies where boiled for 10 minutes in a PCR tube in 10 µl 2 mM NaOH solution of which 2 µl is taken. First conditions according to table 3 where used, using taq Mastermix PCR solution. This yielded no results when put on gel.



Tabel 3 PCR conditions of knock-out PCR check
Repeats Temperature Duration
94 Melting 2:00
30x 94 Melting 1:00
58 Annealing 2:00
72 Elongation 2:00
72 Elongation 10:00
GPA1 A (fw)TCTGCGTATTCTTCCTTGTAGAAAT
GPA1 D (rv) GAATTCGAGATAATACCCTGTCCTT

A second run with conditions according to table 4 was performed. Now colonies where dissolved in 20 µl 2 mM NaOH of which 18 µl was taken (the maximum amount). This yielded no results when put on gel.


Table 4 PCR on pUG73 cassette with Phusion polymerase summary.
Repeats Temperature Duration
94 Melting 2:00
25x 94 Melting 1:00
56 Annealing 1:00
72 Elongation 4:00
72 Elongation 10:00
GPA1 A (fw) TCTGCGTATTCTTCCTTGTAGAAAT
GPA1 D (rv) GAATTCGAGATAATACCCTGTCCTT


Week of 18-07-12

A new PCR kit, phusion polymerase, is tried. PCR conditions and mix are shown in table 5.



Table 5 colony PCR on WT yeast with Phusion polymerase summary.
Repeats Temperature Duration
98 Melting 0:30
25x 98 Melting 0:20
66 Annealing 0:30
72 Elongation 2:30
72 Elongation 10:00
GPA1 A (fw) TCTGCGTATTCTTCCTTGTAGAAAT
GPA1 D (rv) GAATTCGAGATAATACCCTGTCCTT
Phusion mix
5x Phusion buffer10 µl
10 mM dNTP’s1 µl
Primer A5 µl
Primer D5 µl
DNA template (boiled)2 µl
DMSO1.5 µl
Polymerase0.5 µl
H2O25 µl


The PCR product is put on gel, shown in figure 2. Here a band can be observed in lane 3 with a length of ~4000 nt. This PCR product is inconsistent with both the URA gene (~3000 nt) or the original gene (2000 nt). We ordered new primers, using different regions of the gene. Also primers checking the insert where redesigned.


Figure 2 0.8% Agarose gel in TAE, 40 minutes 80 V. In the picture can be seen: 1 DNA smartladder, 2 PCR product with DNA of colony 1, 3 PCR product with DNA of colony 2, 4 PCR product with DNA of colony 3.


Week of 20-07-12


To check whether the new primers were working, colony PCR of wildtype yeast was performed. New primers were ordered for the A and D primers, called confident primers (conf). No product occurred in the negative control, PCR of wildtype yeast with the AB and CD primers was also performed. Before initiation the colonies were boiled for 10 minutes in a PCR tube in 2 mM NaOH solution. Conditions according to table 6 were used, using the Phusion polymerase. No results were seen on gel.


Table 6 Phusion PCR reaction.

Repeats Temperature Duration
98 Melting 0:30
30x 98 Melting 0:10
57 Annealong 0:30
72 Elongation 1:15
72 Elongation 10:00
GPA1 A TCTGCGTATTCTTCCTTGTAGAAAT
KO B CGCCAAGGGTAGAGATCCTAAG
KO C CTTCACGCAGGATGACAGTTC
GPA1 DGAATTCGAGATAATACCCTGTCCTT
GPA1 A conf CGTCCTTCTGCGTATTCTTCC
GPA1 D conf CCGAGTATTTACCAGGGAGAAG


To check whether the problem was due to the treatment we performed a reaction with 18SrDNA Eukaryotic primers: EukAt & Euk516GC which amplifies a region of 563 nt. First a PCR reaction with taq mastermix was performed and pretreatment of the colonies was dissolving in 10 µl 2mM NaOH and taking 3 µl of this mixture as DNA template. 0.25 µl primers were added. The PCR product was put on gel which is shown in figure 3. PCR reactions were performed on wildtype yeast. A band of ~2000 nt can be observed in lane 3 which corresponds to the length of the gene. Further bands of ~500 nt can be observed in lane 2 and 8 which correspond to the expected Euk516GC primer product. Further can be seen that the band in lane 8 is lighter. This PCR reaction was performed on a 15 minute pretreated wildtype colony. Longer pretreatment will make worse the outcome of the reaction.



Table 7 Taq PCR reaction on wildtype yeast and knock-out colonies
Repeats Temperature Duration
94 Melting 4:00
35x 94 Melting 0:30
Annealing 4:00
55 Elongation 0:40
72 Elongation 10:00
GPA1 A (old) TCTGCGTATTCTTCCTTGTAGAAAT
GPA1 D (old) GAATTCGAGATAATACCCTGTCCTT
GPA1 A conf CGTCCTTCTGCGTATTCTTCC
GPA1 D conf CCGAGTATTTACCAGGGAGAAG
KO B CGCCAAGGGTAGAGATCCTAAG
KO C CTTCACGCAGGATGACAGTTC