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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

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:

PICTURE!!

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

Unfortunately there were no bands 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