Plan for the construction of E. coli vector pYPKa_Z_FBA1tp
PCR with primers pfw630 & prv630 and template FBA1_template results in a 643bp PCR product
Primers annealing on template:
5ATAACAATACTGACAGTACTAAA...ACCAAGTAATACATATTCAAA3 ||||||||||||||||||||| tm 42.2 (dbd) 52.2 3TGGTTCATTATGTATAAGTTTaattaat5 5ttaaatATAACAATACTGACAGTACTAAA3 ||||||||||||||||||||||| tm 44.9 (dbd) 51.0 3TATTGTTATGACTGTCATGATTT...TGGTTCATTATGTATAAGTTT5
Suggested PCR programs for Taq polymerase and for Polymerases with DNA binding domain:
Taq (rate 30 nt/s) 35 cycles |643bp 95.0°C |95.0°C | |SantaLucia 1998 |_________|_____ 72.0°C |72.0°C|SaltC 50mM | 03min00s|30s \ ________|______| | | \ 49.0°C/ 0min19s| 5min | | | \_____/ | | | | 30s | |4-12°C
Clone the PCR product in pYPKa digested with ZraI resulting in pYPKa_Z_FBA1tp
Confirm the structure of the pYPKa_Z_FBA1tp using primers 577, 342 and pfw630 in a multiplex PCR reaction.
Expected PCR products sizes from 577, 342 and pfw630 (bp):
pYPKa with insert in correct orientation: 1577, 1409
pYPKa with insert in reverse orientation: 1577, 811
Empty pYPKa clone : 934