Team:Warsaw/Calendar-Main/12 July 2008

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1. Isolation of plasmids from cultures inocluated on previous day (pCACYC177 + OmpA_alpha and pACYC177+OmpA_Z_omega). <br>
1. Isolation of plasmids from cultures inocluated on previous day (pCACYC177 + OmpA_alpha and pACYC177+OmpA_Z_omega). <br>
2. Control digestation of isolated plasmids with BamHI and SacI (we found good clones for both ligations).</p>
2. Control digestation of isolated plasmids with BamHI and SacI (we found good clones for both ligations).</p>
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<p>'''Linked PCR Omega-A'''<br>
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reisolated PCR product PCR-omega - 4 µl<br>
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reisolated PCRT product A-homo - 13.5 µl<br>
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primer OmegaLS - 2 µl<br>
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primer APNot - 2 µl<br>
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Pfu buffer with Mg2+ - 5 µl<br>
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dNTPs - 1 µl<br>
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H2o - 22 µl<br>
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program:<br>
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1. 95&deg;C - 3'
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2. 95&deg;C - 30"
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3. 55&deg;C - 45"
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4. 68&deg;C - 1'
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5. go to step 2 25 x
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6. 68&deg; - 10'
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7. keep in 4&deg;
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gel electrophoresis<br>

Revision as of 16:48, 26 September 2008

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Preparation of constructs with OmpA protein fusions and Cloning of protein Z DNA to OmpA constructs
1. Isolation of plasmids from cultures inocluated on previous day (pCACYC177 + OmpA_alpha and pACYC177+OmpA_Z_omega).
2. Control digestation of isolated plasmids with BamHI and SacI (we found good clones for both ligations).

Linked PCR Omega-A
reisolated PCR product PCR-omega - 4 µl
reisolated PCRT product A-homo - 13.5 µl
primer OmegaLS - 2 µl
primer APNot - 2 µl
Pfu buffer with Mg2+ - 5 µl
dNTPs - 1 µl
H2o - 22 µl
program:
1. 95°C - 3' 2. 95°C - 30" 3. 55°C - 45" 4. 68°C - 1' 5. go to step 2 25 x 6. 68° - 10' 7. keep in 4° gel electrophoresis