Plan for the construction of E. coli vector pYPKa_Z_TEF1tp
PCR with primers pfw579 & prv579 and template TEF1_template results in a 592bp PCR product
Primers annealing on template:
5ACAATGCATACTTTGTAC...TAATCTAAGTTTTAATTACAAA3 |||||||||||||||||||||| tm 39.1 (dbd) 49.3 3ATTAGATTCAAAATTAATGTTTaattaat5 5ttaaatACAATGCATACTTTGTAC3 |||||||||||||||||| tm 41.9 (dbd) 49.5 3TGTTACGTATGAAACATG...ATTAGATTCAAAATTAATGTTT5
Suggested PCR programs for Taq polymerase and for Polymerases with DNA binding domain:
Taq (rate 30 nt/s) 35 cycles |592bp 95.0°C |95.0°C | |SantaLucia 1998 |_________|_____ 72.0°C |72.0°C|SaltC 50mM | 03min00s|30s \ ________|______| | | \ 48.0°C/ 0min17s| 5min | | | \_____/ | | | | 30s | |4-12°C
Clone the PCR product in pYPKa digested with ZraI resulting in pYPKa_Z_TEF1tp
Confirm the structure of the pYPKa_Z_TEF1tp using primers 577, 342 and pfw579 in a multiplex PCR reaction.
Expected PCR products sizes from 577, 342 and pfw579 (bp):
pYPKa with insert in correct orientation: 1526, 1358
pYPKa with insert in reverse orientation: 1526, 760
Empty pYPKa clone : 934