Team:Warsaw/Calendar-Main/9 October 2008

From 2008.igem.org

(Difference between revisions)
Line 7: Line 7:
<h3>Preparation of vector for pT7 constructs</h3>
<h3>Preparation of vector for pT7 constructs</h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
-
 
+
<ol>
-
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmids from cultures inoculated on previous day - <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> without XbaI).</li>
+
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmids from cultures inoculated on previous day - <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> without XbaI.</li>
<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> without XbaI plasmids with XbaI and BamHI (Tango buffer). Gel electrophoresis - proper clones found.</li>
<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> without XbaI plasmids with XbaI and BamHI (Tango buffer). Gel electrophoresis - proper clones found.</li>
-
 
+
</ol>

Revision as of 00:51, 27 October 2008

Gallery Bricks Notebook Team Project Home


Previous day
return to main notebook page
Previous entry
next notebook entry

 



Preparation of vector for pT7 constructs

Michał K.

  1. Isolation of plasmids from cultures inoculated on previous day - pET15b+OmpA_omega without XbaI.
  2. Control digest of isolated pET15b+OmpA_omega without XbaI plasmids with XbaI and BamHI (Tango buffer). Gel electrophoresis - proper clones found.

Preparation of BioBricks

Michał K.

  1. Isolation of plasmids from cultures inoculated on previous day pSB1A3+ AID and pACYC177 + pET15b+OmpA_omega without XbaI).
  2. Control digest of isolated pSB1A3+ AID plasmids with EcoRI and PstI (Orange buffer) and pET15b+OmpA_omega without XbaI plasmids with XbaI and BamHI (Tango buffer). Gel electrophoresis - proper clones found for both ligations.
  3. Digest of pET15b+OmpA_omega without XbaI plasmid with NdeI and SacI (BamHI buffer), dephosphorylation with CIAP
  4. Gel electrophoresis and gel-out of proper band: ??????.
  5. Ligation of digested vector with omega_link (from 30 September) and alpha_link (from 29 September) fragments (1 hr).
  6. PCR on above ligations using pETt7L_XNE/LinP_BS and pETt7L_XNE/AlphaPlinkSac primers separetly (annealing temperature 58 °C; elongation length 120s) to obtain pT7_omega_ and pT7_alpha fragments.
  7. Gel electrophoresis of PCR products and gel-out of proper bands (pT7_omega_ - 600 bp and alpha_link - 800 bp).
  8. Overnight digest of purified PCR products: pT7_omega_ with EcoRI and BcuI (BamHI buffer) and alpha_link with EcoRI and SacI (BamHI buffer).

Piotr

  1. Isolation of plasmid from culture inoculated on previous day pSB2K3 + pLac_OmpA_omega (without EcoRI site).
  2. Control digest of isolated pSB2K3 + pLac_OmpA_omega with EcoRI and PstI (Orange buffer) proper clones found.
  3. Inoculation of colonies from plate with ligation of pMPMT5+AID without EcoRI site to liquid LB + tetracycline.