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)
Three-step| 30 cycles | |SantaLucia 1998
94.0°C |94.0°C | |SaltC 50mM
__________|_____ 72.0°C |72.0°C|
04min00s |30s \ ________|______|
| \ 50.0°C/ 0min21s|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
| \______/ 0min10s|10min |
| 10s | |4-8°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