Cloning of protein Z DNA to pET15b-OmpA-alpha in place of OmpA

Piotr, Antoni

1. Digest of pET15b-OmpA-alpha and Z (in Geneart vector) with NdeI and NotI (Orange buffer).
2. Gel-out of Z (~200 bp band).
3. Overnight ligation of Z into digested pET15b-OmpA-alpha.

Preparation of constructs with OmpA protein fusions

Michał K.

1. Colony PCR on colonies from plates with transformations pACYC177+OmpA_omega and pACYC177+OmpA_alpha. Primers used: OmpaL_N and OmpaP_link.
2. Gel electrophoresis.(Fig. 1., Fig. 2.)
Fig. 1. Colony PCR on primers shown above (vector change from pET15b to pACYC177)
Upper gel:
1. Marker
2-12 PCR from colonies carrying probable Omp_A_alpha
Lower gel
1. Marker
6. negative control
7-12. CR from colonies carrying probable Omp_A_omega

3. Confirmed transformant colonies inoculated to liquid LB with kanamycin.

Preparation of construct pKS with A protein

Michał L., Marcin

1. Gradient PCR (achieving multiple copies of gene coding A protein):
   template DNA pDRIVE-TAPtag - 1 µl
   primer AP+NotI_N - 2 µl
   primer AL+SacI_N - 2 µl
   Pfu buffer with Mg²⁺ - 5 µl
   10 mM dNTPs - 1 µl
   Pfu Turbo polymerase - 0.5 µl
   H2O - 38.5 µl
   
   Program:
   1. 94°C, 3 min
   2. 94°C, 30 sec
   3. 62 to 74°C, 45 sec
   4. 72°C, 45 sec
   5. Repeat of elongation step 25X
   6. 72°C, 10 min
   7. Hold at 4 °C
   


2. Gel electrophoresis of PCR product.
3. Isolation of proper band (470 bp) from the gel.
4. Overnight digest of isolated PCR product and pKS vector with NotI and SacI; to the reaction mix with pKS added 0.5 µl of CIAP.