Cloning of protein Z DNA to OmpA constructs

Michał K.

1. Digest of Geneart_Z and pACYC177+OmpA_omega with SacI and NotI (BamHI buffer), 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 fragment DNA (1 hr).
4. Transformation of E. coli TOP10 strain with ligation.
5. Transformants plating on LB + kanamycin.

Preparation of alpha+A conctruct

Antoni

1. PCR on pKS plasmid containing protein A with AL+link10+homo2 and AP+NotI primers (20 cycles, elongation 40 s, annealing temperature 72°C).
2. PCR on pUC19 plasmid with AlphaL+SacI and AlphaP+link10+homo2 primers (20 cycles, elongation 45 s, annealing temperature 63°C).
   As a result we got two PCR products (alpha-linker and linker-A) wich will be utilized as templates in next PCR.
3. Gel electrophoresis of PCR products and gel-out of proper bands (alpha_linker - 570 bp and linker_A - 470 bp ).
4. PCR on alpha+A PCR products with AlphaL+SacI and AP+NotI primers.
5. Gel electrophoresis reveal lack of proper 1000 bp band.

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 - 2 µl
   primer AL+link10+homo2 - 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 OmegaL+SacI - 2 µl
   primer OmegaP+link10+homo2 - 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