pYPKa_Z_TDH3tp

Plan for the construction of E. coli vector pYPKa_Z_TDH3tp

Step 1 PCR of the insert

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

Step 2 Vector digestion and cloning

Clone the PCR product in pYPKa digested with ZraI resulting in pYPKa_Z_TDH3tp

Step 3 Diagnostic PCR confirmation

Confirm the structure of the pYPKa_Z_TDH3tp using primers 577, 342 and pfw698 in a multiplex PCR reaction.

Expected PCR products sizes from 577, 342 and pfw698 (bp):

pYPKa with insert in correct orientation: 1645, 1477
pYPKa with insert in reverse orientation: 1645, 879
Empty pYPKa clone : 934