Team:Stockholm/12 October 2010




Gel clean up

I loaded samples in an agarose gel 1 % and ran at 80 V.


  • Protein A_TAT_N, _Tra10_N and _LMWP_N
  • Fusion_CPP1, TAT_C ans _CPP3
  • Fusion_TAT_N, _Tra10_N and _LMWP_N

The procedure was according to the description in protocols.

Weight of all samples: 200 mg

200 * 3 = 600 ul QXI

In step 3 I added 10 ul of QIAEXII to each sample.

Polyacrylamide gel

I ran a gel of the purified SOD.His (N terminal) to check if there had been any purification of the protein.

Ladder: PageRuler Unst. Protein Ladder

Arrangement on the gel:




The gel was incubated in coomassie blue staining and destained on shake.

Unfortunately there were no bands representing SOD.His (~17 kDa) on the gel, which must mean that there was too small culture (12 ml) and that the cells have not been lysed properly. I will redo this work but with more start culture and lyse the cells in a more effiecient way.

The polyacrylamide gel was prepared by the SDS-PAGE mixtures described in protocols.


Removal of insertion in BioBrick suffixes

Transformation results

Good colony yield on all plates. A few red colonies discovered on all plates, indicating that some partly digested pSB1C3 vector, still containing the RFP insert, had been excised during gel extraction.

Colony PCR

From 11/10 transformations'

  1. pSB1C3.IgGp A-C
  2. pSB1C3.bFGF A-C
  3. pSB1C3.ProtA A-C
  4. pSB1C3.yCCS A-C
  5. pSB1C3.SOD A-C
  • Standard colony PCR settings
    • 1:15 elongation time

Gel verification

Gel 1. Colony PCR gel verification of cloned with insertion removed.
3 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.
Gel 2. Colony PCR gel verification of cloned with insertion removed.
3 μl λ; 5 μl sample.
λ = O'GeneRuler 50 bp DNA ladder.

Gel 1: 1 % agarose, 140 V
Gel 2: 1 % agarose, 160 V

Expected bands

  1. pSB1C3.IgGp: 1262 bp
  2. pSB1C3.bFGF: 794 bp
  3. pSB1C3.ProtA: 506 bp
  4. pSB1C3.yCCS: 1076 bp
  5. pSB1C3.SOD: 791 bp


  1. All clones correct.
  2. All clones correct.
  3. All clones correct.
  4. Correct size for clone C, weak band for clone B. Clone A probably carrying an RFP insert.
  5. All clones correct.

ON cultures

  • 5 ml LB + Cm 25; 37 °C, 225 rpm
    • 1C, 2C, 3C, 4C, 5C

BL21 transformation and growth test

Testing BL21 cells with/without CPP expression by transforming and inducing with IPTG.

  • 50 μl 0.1 M IPTG
  • 40 μl competent cells
    • pEX.SOD⋅His
    • pEX.nTra10⋅SOD⋅His
    • pEX.nTAT⋅SOD⋅His
    • pEX.nLMWP⋅SOD⋅His


SOD activity assay

  • Kits?
    • Biosite/trevigen 6000sek
    • Biovision/labinova 3000sek, ca 1week delivery time
    • Cayman
    • Cellbiolabs
    • Merch
    • Abcam 3600sek
    • Probior
    • Sigma-Aldrich 3000sek
    • T-cell technology inc.

  • Kit from Sigma-Aldrich
    • sponsored with a very good offer

The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/