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)
Three-step| 30 cycles | |SantaLucia 1998
94.0°C |94.0°C | |SaltC 50mM
__________|_____ 72.0°C |72.0°C|
04min00s |30s \ ________|______|
| \ 48.0°C/ 0min17s|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 \ 50.0°C ________|______|Primer2C 1µM
| \______/ 0min 8s|10min |
| 10s | |4-8°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