Team:Warsaw/Calendar-Main/9 October 2008

From 2008.igem.org

(Difference between revisions)
Line 66: Line 66:
-
 
+
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a></h3>
-
 
+
-
 
+
-
 
+
-
 
+
-
<h3>Preparation of AraC+pBAD+AID</h3>
+
<h4>Piotr</h4>
<h4>Piotr</h4>

Revision as of 20:27, 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.
  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 - 6000 bp.

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K.

  1. Ligation of digested pET15b vector (from Preparation of vector for pT7 constructs) with alpha_linker fragment(from 25 September) (1 hr).
  2. PCR on above ligation using pETt7L_XNE and AlphaPlinkSac primers (annealing temperature 58 °C; elongation length 120s) to obtain alpha_linker under PT7 (BBa_K103019)fragment.
  3. Gel electrophoresis of PCR products and gel-out of proper bands (alpha_linker under PT7 (BBa_K103019) - 800 bp).
  4. Overnight digest of purified PCR product EcoRI and SacI (BamHI buffer).

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

  1. Ligation of digested pET15b vector (from Preparation of vector for pT7 constructs) with omega_linker fragment(from 30 September) (1 hr).
  2. PCR on above ligations using pETt7L_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 120s) to obtain omega_linker under PT7 (BBa_K103020) fragments
  3. Gel electrophoresis of PCR products and gel-out of proper bands (pT7_omega_ - 600 bp).
  4. Overnight digest of purified PCR product with EcoRI and BcuI (BamHI buffer).

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)

Piotr

  1. Isolation of plasmid from culture inoculated on previous day pSB2K3 +BBa_K103018 (without internal EcoRI site).
  2. Control digest of isolated pSB2K3 + BBa_K103018 with EcoRI and PstI (Orange buffer) proper clones found.

Preparation of AID(BBa_K103001)

Michał K.

  1. Isolation of plasmids from cultures inoculated on previous day pSB1A3+ AID(BBa_K103001).
  2. Control digest of isolated plasmids with EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found.

Preparation of AID under pBAD/araC (BBa_K103002)

Piotr

Inoculation of colonies from plate with ligation of pMPMT5+AID without EcoRI site to liquid LB + tetracycline.