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) 35 cycles |1013bp 95.0°C |95.0°C | |SantaLucia 1998 |_________|_____ 72.0°C |72.0°C|SaltC 50mM | 03min00s|30s \ ________|______| | | \ 51.0°C/ 0min30s| 5min | | | \_____/ | | | | 30s | |4-12°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