Team:Warsaw/Calendar-Main/12 July 2008

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(Difference between revisions)
Line 21: Line 21:
program:<br>
program:<br>
-
1. 95&deg;C - 3'
+
1. 95&deg;C - 3'<br>
-
2. 95&deg;C - 30"
+
2. 95&deg;C - 30"<br>
-
3. 55&deg;C - 45"
+
3. 55&deg;C - 45"<br>
-
4. 68&deg;C - 1'
+
4. 68&deg;C - 1'<br>
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5. go to step 2 25 x
+
5. go to step 2 25 x<br>
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6. 68&deg; - 10'
+
6. 68&deg; - 10'<br>
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7. keep in 4&deg;
+
7. keep in 4&deg;<br>
gel electrophoresis<br>
gel electrophoresis<br>

Revision as of 16:49, 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