Plan for the construction of E. coli vector pYPKa_E_PDC1tp
PCR with primers pfw955 & prv955 and template PDC1_template results in a 968bp PCR product
Primers annealing on template:
5AGGGTAGCCTCCCCAT...ACAGTCAAATCAATCAAA3 |||||||||||||||||| tm 41.1 (dbd) 52.7 3TGTCAGTTTAGTTAGTTTaattaat5 5ttaaatAGGGTAGCCTCCCCAT3 |||||||||||||||| tm 49.1 (dbd) 61.3 3TCCCATCGGAGGGGTA...TGTCAGTTTAGTTAGTTT5
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 \ ________|______| | \ 50.0°C/ 0min29s|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 \ 53.0°C ________|______|Primer2C 1µM | \______/ 0min14s|10min | | 10s | |4-8°C
Clone the PCR product in pYPKa digested with EcoRV resulting in pYPKa_E_PDC1tp
Confirm the structure of the pYPKa_E_PDC1tp using primers 568, 342 and pfw955 in a multiplex PCR reaction.
Expected PCR products sizes from 568, 342 and pfw955 (bp):
pYPKa with insert in correct orientation: 1684, 1653
pYPKa with insert in reverse orientation: 1684, 999
Empty pYPKa clone : 716