pYPKa_E_TDH3tp

Plan for the construction of E. coli vector pYPKa_E_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) 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

Step 2 Vector digestion and cloning

Clone the PCR product in pYPKa digested with EcoRV resulting in pYPKa_E_TDH3tp

Step 3 Diagnostic PCR confirmation

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