Plan for the construction of E. coli vector pYPKa_A_ScTAL1
PCR with primers pfw1008 & prv1008 and template ScTAL1_template results in a 1010bp PCR product
Primers annealing on template:
5ATGTCTGAACCAGCTC...AAGAAAGTTACCGCTTAA3 |||||||||||||||||| tm 42.9 (dbd) 53.4 3TTCTTTCAATGGCGAATT5 5aaATGTCTGAACCAGCTC3 |||||||||||||||| tm 44.1 (dbd) 52.9 3TACAGACTTGGTCGAG...TTCTTTCAATGGCGAATT5
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 \ ________|______| | \ 52.0°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 \ 53.0°C ________|______|Primer2C 1µM | \______/ 0min15s|10min | | 10s | |4-8°C
Clone the PCR product in pYPKa digested with AjiI resulting in pYPKa_A_ScTAL1
Confirm the structure of the pYPKa_A_ScTAL1 using primers 468, 342 and pfw1008 in a multiplex PCR reaction.
Expected PCR products sizes from 468, 342 and pfw1008 (bp):
pYPKa with insert in correct orientation: 1776, 1726
pYPKa with insert in reverse orientation: 1776, 1060
Empty pYPKa clone : 766