Linearize pYPKpw with EcoRV resulting in the linearized vector.
Carry out a PCR with primers 577, 567 and template pYPKa_Z_PGItp resulting in
the PCR product 1231bp_PCR_prod
5GTTCTGATCCTCGAGCATCTTAAGAATTC...CTCACTAGTGACCTGCAGCCGAC3 ||||||||||||||||||||||| tm 59.0 (dbd) 70.7 3gagtgatcactggacgtcggcTG5 5gttctgatcctcgagcatcttaagaattc3 ||||||||||||||||||||||||||||| tm 56.1 (dbd) 69.4 3CAAGACTAGGAGCTCGTAGAATTCTTAAG...GAGTGATCACTGGACGTCGGCTG5 Taq (rate 30 nt/s) Three-step| 30 cycles | |SantaLucia 1998 94.0°C |94.0°C | |SaltC 50mM __________|_____ 72.0°C |72.0°C| 04min00s |30s \ ________|______| | \ 56.0°C/ 0min36s|10min | | \_____/ | | | 30s | |4-8°C Pfu-Sso7d (rate 15s/kb) Two-step| 30 cycles | |Breslauer1986,SantaLucia1998 98.0°C |98.0C | |SaltC 50mM _____ __|_____ | |Primer1C 1000µM 00min30s|10s \ 72.0°C|72.0°C|Primer2C <bound method Amplicon.rc of Amplicon(1231)>µM | \_______|______| | 0min18s|10min |4-8°C
Carry out a PCR with primers 468, 467 and template pYPKa_A_ScXKS1 resulting in
the PCR product 1892bp_PCR_prod
5GTCGAGGAACGCCAGGTTGCCCACT...TCTGTGCAGACAAACGCATCAGGAT3 ||||||||||||||||||||||||| tm 59.0 (dbd) 73.8 3agacacgtctgtttgcgtagtcctaAATTTA5 5gtcgaggaacgccaggttgcccact3 ||||||||||||||||||||||||| tm 64.8 (dbd) 79.7 3CAGCTCCTTGCGGTCCAACGGGTGA...AGACACGTCTGTTTGCGTAGTCCTA5 Taq (rate 30 nt/s) Three-step| 30 cycles | |SantaLucia 1998 94.0°C |94.0°C | |SaltC 50mM __________|_____ 72.0°C |72.0°C| 04min00s |30s \ ________|______| | \ 57.0°C/ 0min56s|10min | | \_____/ | | | 30s | |4-8°C Pfu-Sso7d (rate 15s/kb) Two-step| 30 cycles | |Breslauer1986,SantaLucia1998 98.0°C |98.0C | |SaltC 50mM _____ __|_____ | |Primer1C 1000µM 00min30s|10s \ 72.0°C|72.0°C|Primer2C <bound method Amplicon.rc of Amplicon(1892)>µM | \_______|______| | 0min28s|10min |4-8°C
Carry out a PCR with primers 568, 578 and template pYPKa_E_FBA1tp resulting in
the PCR product 969bp_PCR_prod
5GTGCCATCTGTGCAGACAAACG...ACTTATGAATGTGGCAATGAGACAAGAAC3 ||||||||||||||||||||||||||||| tm 56.5 (dbd) 69.5 3tgaatacttacaccgttactctgttcttg5 5GTGCcatctgtgcagacaaacg3 |||||||||||||||||||||| tm 57.1 (dbd) 71.5 3CACGGTAGACACGTCTGTTTGC...TGAATACTTACACCGTTACTCTGTTCTTG5 Taq (rate 30 nt/s) Three-step| 30 cycles | |SantaLucia 1998 94.0°C |94.0°C | |SaltC 50mM __________|_____ 72.0°C |72.0°C| 04min00s |30s \ ________|______| | \ 56.0°C/ 0min29s|10min | | \_____/ | | | 30s | |4-8°C Pfu-Sso7d (rate 15s/kb) Two-step| 30 cycles | |Breslauer1986,SantaLucia1998 98.0°C |98.0C | |SaltC 50mM _____ __|_____ | |Primer1C 1000µM 00min30s|10s \ 72.0°C|72.0°C|Primer2C <bound method Amplicon.rc of Amplicon(969)>µM | \_______|______| | 0min14s|10min |4-8°C
Mix the four linear DNA fragments and transform a Saccharomyces cerevisiae ura3 mutant with the mixture. The fragments will be assembled by in-vivo homologous recombination:
-|pYPKpw|124 | \/ | /\ | 124|1231bp_PCR_prod|50 | \/ | /\ | 50|1892bp_PCR_prod|37 | \/ | /\ | 37|969bp_PCR_prod|242 | \/ | /\ | 242- | | ---------------------------------------------------------------------
PCR using primers 577 & 467
PCR products (bp)
Correct : 3073
Missing first tp : 2060
Missing gene : 1268
Missing both : 255
PCR using primers 468 & 578
PCR products (bp)
Correct : 2824
Missing gene : 1019
Missing last tp : 2181
Missing both : 376