Team:Warsaw/Calendar-Main/12 July 2008

From 2008.igem.org

(Difference between revisions)
Line 12: Line 12:
reisolated PCR product PCR-omega - 4 µl<br>
reisolated PCR product PCR-omega - 4 µl<br>
-
reisolated PCRT product A-homo - 13.5 µl<br>
+
reisolated PCR product A-homo - 13.5 µl<br>
-
primer OmegaLS - 2 µl<br>
+
primer <html>
-
primer APNot - 2 µl<br>
+
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaLS_N">OmegaLS_N</a> - 2 µl<br>
 +
primer <html>
 +
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#APNot_N">APNot_N</a>APNot - 2 µl<br>
Pfu buffer with Mg2+ - 5 µl<br>
Pfu buffer with Mg2+ - 5 µl<br>
dNTPs - 1 µl<br>
dNTPs - 1 µl<br>

Revision as of 16:58, 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 PCR product A-homo - 13.5 µl
primer OmegaLS_N - 2 µl
primer APNot_NAPNot - 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
{{WarNotebookEnd}}