Preparation of constructs with OmpA protein fusions
1. Digestion of confirmed clones of pET15b+OmpA_alpha and pET15b+OmpA_omega with NdeI and BamHI
2. Digestion of pACYC177 plasmid with NdeI and BamHI, dephosphorylation with CIAP
3. Gel electrophoresis and digested plasmids and gel-out of proper bands
4. Overnight ligation of pACYC177 and OmpA_alpha
5. Overnight ligation of pACYC177 and OmpA_omega
Preparation of construct pKS with A protein
1. Gradient PCR:
template DNA pDRIVE-TAPtag - 1 µl
primer APNot - 2 µl
primer ALSac - 2µl
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°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 (470bp) from the gel
4. Overnight digestion of isolated PCR product with NotI and SacI
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