Preparation of alfa+A conctruct

Antoni

1. Protein A PCR on pKS+A
2. PCR on alpha
3. Gel electrophoresis
4. Gel-out
5. PCR on alpha+A

Cloning of protein Z DNA to OmpA constructs

1. Digest of Geneart_Z and pACYC177+OmpA_omega with SacI and NotI (pACYC177 was also dephosphorylated with CIAP)(3 hr).
2. Gel electrophoresis and gel-out of proper bands 220 bp (for Geneart_Z lane)and 4050 bp (pACYC177+OmpA_omega lane).
3. Ligation of pACYC177+OmpA_omega and Z (1 hr).
4. Transformation of E. coli TOP10 strain with ligation.
5. Transformants plating on LB + kanamycin.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin:

We had to start form scratch with this one.

1. PCR A in 50 µl
   template DNA - pKS-A4 1 µl
   primer AP+NotI_N - 2 µl
   primer AL+link10+homo2_N - 2 µl
   Pfu polymerase buffer + Mg²⁺ (from Fermentas) - 5 µl
   dNTPs - 1 µl
   Pfu turbo - 0.5 µl
   H2o - 38.5 µl
   
   Program:
   
   1. 95°C 3'
   2. 95°C 30"
   3. 62°C 45"
   4. 72°C 45"
   5. 72°C 10'
   6. keeping in 4°C
2. PCR omega in 50 µl
   template DNA - pUC19 1 µl
   primer OmegaLS - 2 µl
   primer AOmegaPli - 2 µl
   Pfu polymerase buffer + Mg²⁺ (from Fermentas) - 5 µl
   dNTPs - 1 µl
   Pfu turbo - 0.5 µl
   H2o - 38.5 µl
   Program:
   
   1. 95°C 3'
   2. 95°C 30"
   3. 62°C 45"
   4. 72°C 45"
   5. 72°C 10'
   6. keeping in 4°C
   25 cycles
3. Gel electrophoresis
4. Reisolation from agarose gel