Team:TU-Delft/results

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