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From 2010.igem.org

Contents 1 Nina 1.1 Gel clean up 1.2 Polyacrylamide gel 2 Andreas 2.1 Removal of insertion in BioBrick suffixes 2.1.1 Transformation results 2.1.2 Colony PCR 2.1.3 Gel verification 2.1.4 ON cultures 2.2 BL21 transformation and growth test 3 Mimmi 3.1 SOD activity assay

Nina

Gel clean up

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

Samples:

• 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:

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.

Andreas

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: 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

Results

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

Mimmi

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