Plan for the construction of E. coli vector pYPKa_Z_PGItp
PCR with primers pfw1000 & prv1000 and template PGI_template results in a 1013bp PCR product
Primers annealing on template:
5AATTCAGTTTTCTGACTGA...CAAGATACCAGCCTAAAA3 |||||||||||||||||| tm 43.0 (dbd) 54.5 3GTTCTATGGTCGGATTTTaattaat5 5ttaaatAATTCAGTTTTCTGACTGA3 ||||||||||||||||||| tm 43.6 (dbd) 53.8 3TTAAGTCAAAAGACTGACT...GTTCTATGGTCGGATTTT5
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 \ ________|______| | \ 51.0°C/ 0min30s|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 \ 54.0°C ________|______|Primer2C 1µM | \______/ 0min15s|10min | | 10s | |4-8°C
Clone the PCR product in pYPKa digested with ZraI resulting in pYPKa_Z_PGItp
Confirm the structure of the pYPKa_Z_PGItp using primers 577, 342 and pfw1000 in a multiplex PCR reaction.
Expected PCR products sizes from 577, 342 and pfw1000 (bp):
pYPKa with insert in correct orientation: 1947, 1779
pYPKa with insert in reverse orientation: 1947, 1181
Empty pYPKa clone : 934