Plan for the construction of E. coli vector pYPKa_Z_TDH3tp
PCR with primers pfw698 & prv698 and template TDH3_template results in a 711bp PCR product
Primers annealing on template:
5ATAAAAAACACGCTTTTTC...AACACACATAAACAAACAAA3 |||||||||||||||||||| tm 44.1 (dbd) 54.7 3TTGTGTGTATTTGTTTGTTTaattaat5 5ttaaatATAAAAAACACGCTTTTTC3 ||||||||||||||||||| tm 42.7 (dbd) 55.3 3TATTTTTTGTGCGAAAAAG...TTGTGTGTATTTGTTTGTTT5
Suggested PCR programs for Taq polymerase and for Polymerases with DNA binding domain:
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 \ ________|______| | \ 50.0°C/ 0min21s|10min | | \_____/ | | | 30s | |4-8°C Pfu-Sso7d (rate 15s/kb) Three-step| 30 cycles | |Breslauer1986,SantaLucia1998 98.0°C |98.0°C | |SaltC 50mM __________|_____ 72.0°C |72.0°C|Primer1C 1µM 00min30s |10s \ 55.0°C ________|______|Primer2C 1µM | \______/ 0min10s|10min | | 10s | |4-8°C
Clone the PCR product in pYPKa digested with ZraI resulting in pYPKa_Z_TDH3tp
Confirm the structure of the pYPKa_Z_TDH3tp using primers 577, 342 and pfw698 in a multiplex PCR reaction.
Expected PCR products sizes from 577, 342 and pfw698 (bp):
pYPKa with insert in correct orientation: 1645, 1477
pYPKa with insert in reverse orientation: 1645, 879
Empty pYPKa clone : 934