Preparation of constructs with OmpA protein fusions
Piotr:

1. Digest of confirmed clones of pET15b+OmpA_alpha and pET15b+OmpA_omega with NdeI and BamHI.
2. Digest of pACYC177 plasmid with NdeI and BamHI, dephosphorylation with CIAP.
3. Gel electrophoresis of digested plasmids and gel-out of proper bands.
4. Electrophoresis of gel-out products.
5. Overnight ligation of pACYC177 and OmpA_alpha.
6. Overnight ligation of pACYC177 and OmpA_omega.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin:

We have just received corrected version of omegaP-link10-homo2 primer. Let's hope this time everything will be fine. We are repeating the PCRs:

PCR program for linker-A and omega-linker

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.