Plan for the construction of E. coli vector pYPKa_A_SsXYL2
PCR with primers pfw1092 & prv1092 and template SsXYL2_template results in a 1094bp PCR product
Primers annealing on template:
5ATGACTGCTAACCCTTC...TGACGGCCCTGAGTAA3
|||||||||||||||| tm 48.0 (dbd) 60.8
3ACTGCCGGGACTCATT5
5aaATGACTGCTAACCCTTC3
||||||||||||||||| tm 44.5 (dbd) 54.0
3TACTGACGATTGGGAAG...ACTGCCGGGACTCATT5
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 \ ________|______|
| \ 55.0°C/ 0min32s|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
| \______/ 0min16s|10min |
| 10s | |4-8°C
Clone the PCR product in pYPKa digested with AjiI resulting in pYPKa_A_SsXYL2
Confirm the structure of the pYPKa_A_SsXYL2 using primers 468, 342 and pfw1092 in a multiplex PCR reaction.
Expected PCR products sizes from 468, 342 and pfw1092 (bp):
pYPKa with insert in correct orientation: 1860, 1810
pYPKa with insert in reverse orientation: 1860, 1144
Empty pYPKa clone : 766