Step 1 PCR of the insert

PCR with primers pfw630 & prv630 and template FBA1_template results in a 643bp PCR product

Primers annealing on template:

      5ATAACAATACTGACAGTACTAAA...ACCAAGTAATACATATTCAAA3
                                 ||||||||||||||||||||| tm 42.2 (dbd) 52.2
                                3TGGTTCATTATGTATAAGTTTaattaat5
5ttaaatATAACAATACTGACAGTACTAAA3
       ||||||||||||||||||||||| tm 44.9 (dbd) 51.0
      3TATTGTTATGACTGTCATGATTT...TGGTTCATTATGTATAAGTTT5

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  \         ________|______|
          |      \ 49.0°C/ 0min19s|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 9s|10min |
          |        10s           |      |4-8°C

Step 2 Vector digestion and cloning

Clone the PCR product in pYPKa digested with ZraI resulting in pYPKa_Z_FBA1tp

Step 3 Diagnostic PCR confirmation

Confirm the structure of the pYPKa_Z_FBA1tp using primers 577, 342 and pfw630 in a multiplex PCR reaction.

Expected PCR products sizes from 577, 342 and pfw630 (bp):

pYPKa with insert in correct orientation: 1577, 1409
pYPKa with insert in reverse orientation: 1577, 811
Empty pYPKa clone : 934