Preparation of constructs with OmpA protein fusions

1. Colony PCR on colonies from plates with transformations pACYC177+OmpA_omega and pACYC177+OmpA_alpha Primers used: OmpaL_N and OmpaP_link
2. 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 <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI_N">AP+NotI_N</a></html> - 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.

Preparation of construct pKS with A protein

1. Colony PCR on colonies from plates with transformations pKS-A
   Primers used: AL+SacI and AP+NotI
2. Confirmed transformant colonies inoculated to liquid LB with ampicillin