Team:EPF-Lausanne/Notebook/21 September 2012

From 2012.igem.org



Contents

CHO and HEK passaging

Made 4 tubes of HEK seed and of CHO seed (each about 1.5 mio/ml) for the Sunday transfection.


Silver Staining result

Protocol: Silver Staining


Gel making

Gel Ingredients (choose percentage according to the size of the protein)

4-40 kDA 20%
12-45 kDA 15%
10-70 kDA 12.5%
15-100 kDA 10%
25-200 kDA 8%


Separating gel
Gel percentage 7.5 %
30% Polyacrylamide 10 mL
1.5M Tris (pH 8.8) 10 mL
10% Ammonium persulfate 0.4 mL
10% SDS 0.4 mL
TEMED 0.038 mL
H2O 19.2 mL
Total volume 40 mL
Stacking gel
Gel percentage 5 %
30% Polyacrylamide 1.36 mL
1M Tris (pH 6.8) 1 mL
10% Ammonium persulfate 0.08 mL
10% SDS 0.08 mL
TEMED 0.008 mL
H2O 5.44 mL
Total volume 8 mL


Team-EPF-Lausanne ssp.jpg

  • Loading Plan
  • Lane 1: Ladder
  • Lane 2: Empty well
  • Lane 3: 10microL of VP16 only
  • Lane 4: 25microL of VP16 only
  • Lane 5: Nontransfected CHO cell - High concentration (5million)
  • Lane 6: Sample 1 48h of expression
  • Lane 7: Sample 2 48h of expression
  • Lane 8: Sample 1 72h of expression
  • Lane 9: Sample 2 72h of expression
  • Lane 10: Nontransfected CHO cell - Same concentration as samples


Team-EPF-Lausanne 21.sep.12 ssr.jpg

Result: The lane 9 (Lovtap-VP16 transfected CHO cell) and lane 10 (non-transfected CHO cell) have intensity difference around 30~35kDa area.




  • Loading Plan
  • Lane 1: Ladder
  • Lane 2: Empty well
  • Lane 3: 10 µl of VP16 only
  • Lane 4: 25 µl of VP16 only
  • Lane 5: Nontransfected CHO cells - high concentration (5 million)
  • Lane 6: Sample 1 48h of expression
  • Lane 7: Sample 2 48h of expression
  • Lane 8: Sample 1 72h of expression
  • Lane 9: Sample 2 72h of expression
  • Lane 10: Nontransfected CHO cells - same concentration as samples


Team-EPF-Lausanne 21.sep.12 ssr.jpg

Result: Lane 9 (LovTAP-VP16 transfected CHO cell) and lane 10 (non-transfected CHO cells) have a (faint) intensity difference around 30~35kDa area. This, however, cannot be considered as a significative result.