Plan for the construction of E. coli vector pYPKa_E_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 EcoRV resulting in pYPKa_E_PGItp
Confirm the structure of the pYPKa_E_PGItp using primers 568, 342 and pfw1000 in a multiplex PCR reaction.
Expected PCR products sizes from 568, 342 and pfw1000 (bp):
pYPKa with insert in correct orientation: 1729, 1698
pYPKa with insert in reverse orientation: 1729, 1044
Empty pYPKa clone : 716