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) 35 cycles |1094bp 95.0°C |95.0°C | |SantaLucia 1998 |_________|_____ 72.0°C |72.0°C|SaltC 50mM | 03min00s|30s \ ________|______| | | \ 55.0°C/ 0min32s| 5min | | | \_____/ | | | | 30s | |4-12°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