Step 1 PCR of the insert

PCR with primers pfw579 & prv579 and template TEF1_template results in a 592bp PCR product

Primers annealing on template:

      5ACAATGCATACTTTGTACG...AATCTAATCTAAGTTTTAATTACAAA3
                             |||||||||||||||||||||||||| tm 44.0 (dbd) 54.3
                            3TTAGATTAGATTCAAAATTAATGTTTaattaat5
5ttaaatACAATGCATACTTTGTACG3
       ||||||||||||||||||| tm 45.6 (dbd) 55.3
      3TGTTACGTATGAAACATGC...TTAGATTAGATTCAAAATTAATGTTT5

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/ 0min17s|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
          |      \______/ 0min 8s|10min |
          |        10s           |      |4-8°C

Step 2 Vector digestion and cloning

Clone the PCR product in pYPKa digested with ZraI resulting in pYPKa_Z_TEF1tp

Step 3 Diagnostic PCR confirmation

Confirm the structure of the pYPKa_Z_TEF1tp using primers 577, 342 and pfw579 in a multiplex PCR reaction.

Expected PCR products sizes from 577, 342 and pfw579 (bp):

pYPKa with insert in correct orientation: 1526, 1358
pYPKa with insert in reverse orientation: 1526, 760
Empty pYPKa clone : 934