Team:TU-Delft/results

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<h6>Tabel 1 PCR conditions of knock-out PCR check</h6>
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<td><b>GPA1 A (fw)</b>TCTGCGTATTCTTCCTTGTAGAAAT</td>
<td><b>GPA1 A (fw)</b>TCTGCGTATTCTTCCTTGTAGAAAT</td>

Revision as of 10:23, 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 1 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