Plan for the construction of E. coli vector pYPKa_E_TDH3tp
PCR with primers pfw698 & prv698 and template TDH3_template results in a 711bp PCR product
Primers annealing on template:
5ATAAAAAACACGCTTTTTC...AACACACATAAACAAACAAA3 |||||||||||||||||||| tm 44.1 (dbd) 54.7 3TTGTGTGTATTTGTTTGTTTaattaat5 5ttaaatATAAAAAACACGCTTTTTC3 ||||||||||||||||||| tm 42.7 (dbd) 55.3 3TATTTTTTGTGCGAAAAAG...TTGTGTGTATTTGTTTGTTT5
Suggested PCR programs for Taq polymerase and for Polymerases with DNA binding domain:
Taq (rate 30 nt/s) 35 cycles |711bp 95.0°C |95.0°C | |SantaLucia 1998 |_________|_____ 72.0°C |72.0°C|SaltC 50mM | 03min00s|30s \ ________|______| | | \ 50.0°C/ 0min21s| 5min | | | \_____/ | | | | 30s | |4-12°C
Clone the PCR product in pYPKa digested with EcoRV resulting in pYPKa_E_TDH3tp
Confirm the structure of the pYPKa_E_TDH3tp using primers 568, 342 and pfw698 in a multiplex PCR reaction.
Expected PCR products sizes from 568, 342 and pfw698 (bp):
pYPKa with insert in correct orientation: 1427, 1396
pYPKa with insert in reverse orientation: 1427, 742
Empty pYPKa clone : 716