Niveles de acceso y códigos de seguridad Nota Nota

2.2.4.1 Generation of 9myc C-terminally tagged Rif2

The RIF2-9MYC strain was created by amplifying the 9MYC-KAN cassette by PCR from the plasmid pBL327 by using the oligonucleotides oAL22 and oAL23, which were designed in accordance to (Janke et al., 2004). The PCR reaction was composed of 100 ng pBL327, 25 µl Phusion HF 2x Mastermix, 0.64 µl of each 5 µM oligonucleotide and water to 50 µl. The PCR was performed as follows: 98°C 30 sec, 98°C 10 sec, 72°C 30 sec, 72°C 1 min (to step 2 x 34 times), 72°C 10 min. Correct PCR product length was verified by agarose gel, and subsequently 10 µl of cassette were transformed in yBL7 and plated on YPD + KAN plates, which were grown at 30°C for 3 days. Colonies that grew were restreaked on YPD + KAN to exclude false positives, and those which could re-grow were verified for correct integration by PCR on genomic DNA, which was extracted with Puregene Yeast/Bact. Kit B. The PCR to verify correct construct integration and for subsequent sequencing was performed with the oligonucleotides oAL24 and oBL29, and the reaction was composed of 200 ng genomic DNA, 5 µl of each 5 µM oligonucleotide, 12.5 µl Phusion HF 2X Mastermix and water to 25 µl. The PCR was performed as follows: 98°C 30 sec, 98°C 10 sec, 64°C 30 sec, 72°C 1 min 30 sec (to step 2 x 34 times), 72°C 10 min. Correct product length was verified by agarose gel and sequencing was performed on the PCR product purified with QIAquick PCR Purification Kit, using oAL24 and oBL29. Expression of the tagged protein was confirmed by western blot.

2.2.4.2 Generation of 9myc C-terminally tagged Rif1

The RIF1-9MYC strain was created by amplifying by PCR the 9MYC-NAT cassette from the plasmid pBL334 by using the oligonucleotides oLP1 and oLP2, which were designed in accordance to (Janke et al., 2004). The PCR reaction was composed of 100 ng pBL334, 25 µl Phusion HF 2x Mastermix, 0.64 µl of each 5 µM oligonucleotide, 3% DMSO and water to 50 µl. The PCR was performed as follows: 98°C 30 sec, 98°C 10 sec, 62°C 30 sec, 72°C 1 min 10 sec (to step 2 x 34 times), 72°C 10 min. Correct PCR product length was verified by agarose gel and subsequently 10 µl of cassette were transformed in yBL7 and plated on YPD + NAT plates, which were grown at 30°C for 3 days. Grown colonies were restreaked on YPD + NAT to exclude false positives, and those which could re-grow were verified for correct integration by PCR on genomic DNA, which was extracted with Puregene Yeast/Bact. Kit B. The PCR to verify correct construct integration and for subsequent sequencing was performed with the oligonucleotides oLP3 and oBL29, and was composed of 200 ng genomic DNA, 5 µl of each 2.5 µM oligonucleotide, 12.5 µl Phusion HF 2X Mastermix and water to 25 µl. The PCR was performed as follows: 98°C 30 sec, 98°C 10 sec, 65°C 30 sec, 72°C 1 min (to step 2 x 34 times), 72°C 5 min. Correct PCR product length was verified by agarose gel and sequencing was performed on the PCR product purified with QIAquick PCR Purification Kit, using oLP3 and oBL29. Expression of the tagged protein was confirmed by western blot.

2.2.4.3 Generation of S- and G2-RNH1-TAP alleles

The S- and G2-RNH1-TAP alleles were created by amplifying the S cassette (containing the CLB6 promoter, the first 585 bp of CLB6 and the NAT resistance cassette) from the plasmid pBL491 with the oligonucleotides oAL47 and oAL48, or the G2 cassette (containing the CLB2 promoter, the first 540 bp of CLB2 and the NAT resistance cassette) from the plasmid pBL492 with the oligonucleotides oAL47 and oAL49. The PCR reactions were composed of 100 ng plasmid DNA, 0.64 µl of each 5 µM oligonucleotide, 25 µl Phusion HF 2x Mastermix and water to 50 µl. The PCRs were performed as in (Janke et al., 2004): 97°C 3 min, 97°C 1 min, 54°C 30 sec, 72°C 2 min 40 sec (to step 2 x 10 times), 97°C 1 min, 54°C 30 sec, 72°C 2 min 40 sec (to step 5 x 20 times, extending the elongation time of 20 sec per cycle). Correct cassette lengths were verified by agarose gel, and

ySLG252 was transformed independently with 7.5 µl of each cassette, and plated on YPD + NAT plates, which were grown at 30°C for 3 days. Grown colonies were restreaked on YPD + NAT to exclude false positives, and those which could re-grow were verified for correct integration by PCR on genomic DNA, with the oligonucleotide pairs oAL53 + oAL54 and oAL53 + oBL29. The PCR reactions were composed of: 2 µl of cells (boiled 10 min in 0.02 N NaOH), 2 µl of each 5 µM oligonucleotide, 10 µl Taq 2x Mastermix and water to 20 µl. The PCRs were performed as follows: 98°C 3 min, 98°C 10 sec, 52°C 30 sec, 68°C 4 min (to step 2 x 34 times), 68°C 5 min. Correct product lengths were verified on an agarose gel, and correct expression of RNH1 in the desired cell cycle phases was assayed by western blot (Figure 15).

2.2.4.4 Generation of the S-RNH202-TAP allele

The S-RNH202-TAP allele was created by amplifying the S cassette (containing the CLB6 promoter, the first 585 bp of CLB6 and the NAT resistance cassette) from the plasmid pBL491 with the oligonucleotides oAL61 and oAL62. The PCR reaction was composed of 100 ng pBL491, 0.64 µl of each 5 µM oligonucleotide, 25 µl Phusion HF 2x Mastermix and water to 50 µl. The PCR program was as in (Janke et al., 2004): 97°C 3 min, 97°C 1 min, 54°C 30 sec, 72°C 2 min 40 sec (to step 2 x 10 times), 97°C 1 min, 54°C 30 sec, 72°C 2 min 40 sec (to step 5 x 20 times, extending the elongation time of 20 sec per cycle). Correct cassette length was verified by agarose gel, and yAL350 was transformed with 10 µl cassette and plated on YPD + NAT plates, which were grown at 30°C for 3 days. Grown colonies were restreaked on YPD + NAT to exclude false positives, and those which could re-grow were verified for correct integration by PCR on genomic DNA with the oligonucleotide pairs yAL64 + yAL65 and yAL64 + oBL29. The PCR reactions were composed of: 2 µl cells (boiled 10 min in 0.02 N NaOH), 2 µl of each oligonucleotide, 10 µl Taq 2x Mastermix and water to 20 µl. The PCRs were performed as follows: 98°C 3 min, 98°C 10 sec, 52°C 30 sec, 68°C 4min (to step 2 x 34 times), 68°C 5 min. Correct product length was verified on an agarose gel, and correct expression of RNH1 in the desired cell cycle phase was assayed by western blot (Figure 19).

2.2.5 Chromatin Immunoprecipitation (ChIP) and DNA-RNA Immunoprecipitation