Plan for the construction of E. coli vector pYPKa_A_ScXKS1
PCR with primers pfw1803 & prv1803 and template ScXKS1_template results in a 1805bp PCR product
Primers annealing on template:
5ATGTTGTGTTCAGTAATTCA...TGGAAAAGACTCTCATCTAA3 |||||||||||||||||||| tm 45.0 (dbd) 55.7 3ACCTTTTCTGAGAGTAGATT5 5aaATGTTGTGTTCAGTAATTCA3 |||||||||||||||||||| tm 44.8 (dbd) 54.6 3TACAACACAAGTCATTAAGT...ACCTTTTCTGAGAGTAGATT5
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 \ ________|______| | \ 52.0°C/ 0min54s|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 | \______/ 0min27s|10min | | 10s | |4-8°C
Clone the PCR product in pYPKa digested with AjiI resulting in pYPKa_A_ScXKS1
Confirm the structure of the pYPKa_A_ScXKS1 using primers 468, 342 and pfw1803 in a multiplex PCR reaction.
Expected PCR products sizes from 468, 342 and pfw1803 (bp):
pYPKa with insert in correct orientation: 2571, 2521
pYPKa with insert in reverse orientation: 2571, 1855
Empty pYPKa clone : 766