Plan for the construction of E. coli vector pYPKa_A_SsXYL1
PCR with primers pfw957 & prv957 and template SsXYL1_template results in a 959bp PCR product
Primers annealing on template:
5ATGCCTTCTATTAAGTTGAA...AAGATTCCTATCTTCGTCTAA3
||||||||||||||||||||| tm 45.3 (dbd) 55.5
3TTCTAAGGATAGAAGCAGATT5
5aaATGCCTTCTATTAAGTTGAA3
|||||||||||||||||||| tm 43.8 (dbd) 54.9
3TACGGAAGATAATTCAACTT...TTCTAAGGATAGAAGCAGATT5
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 \ ________|______|
| \ 54.0°C/ 0min28s|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 \ 55.0°C ________|______|Primer2C 1µM
| \______/ 0min14s|10min |
| 10s | |4-8°C
Clone the PCR product in pYPKa digested with AjiI resulting in pYPKa_A_SsXYL1
Confirm the structure of the pYPKa_A_SsXYL1 using primers 468, 342 and pfw957 in a multiplex PCR reaction.
Expected PCR products sizes from 468, 342 and pfw957 (bp):
pYPKa with insert in correct orientation: 1725, 1675
pYPKa with insert in reverse orientation: 1725, 1009
Empty pYPKa clone : 766