Team:Stockholm/25 August 2010

From 2010.igem.org


Contents

Andreas

Transfer of MITF to pSB1C3

Transformation results

From 24/8 Due to late transformation 24/8, colonies didn't turn red until very late in the afternoon, revealing only two white colonies. These will be picked for colony PCR tomorrow.

Construction of His⋅SOD fusions

Plasmid prep

From 24/8 ON cultures

  • E.Z.N.A. Plasmid Mini Prep kit
  • Elution volume: 50 μl
  • Samples eluted twice to increase plasmid yield
DNA concentrations
  Original concentration Conc. after evaporation
Sample ng/μl A260/A280 ng/μl A260/A280
pSB1K3.His⋅SOD 1 52.52 1.73 127.1 1.84
pSB1K3.His⋅SOD 3 41.13 1.74 101.8 1.84
pSB1K3.His⋅yCCS 1 67.48 1.79 133.2 1.86
pSB1K3.His⋅yCCS 2 37.63 1.87 146.7 1.88

New ON cultures of pSB1K3.His⋅SOD 1 and pSB1K3.His⋅yCCS 2 were set for plasmid prep.

  • 5 ml LB + Cm 25; 37 °C, 250 rpm.

Glycerol stocks

  • pMA.His: pMA.His 25/8
  • pSB1K3.yCCS⋅His 2: pK y⋅H2 25/8
  • pSB1K3.yCCS⋅His 3: pK y⋅H3 25/8
  • pSB1K3.His⋅SOD 1: pK H⋅S1 25/8
  • pSB1K3.His⋅SOD 3: pK H⋅S3 25/8
  • pSB1K3.His⋅yCCS 1: pK H⋅y1 25/8
  • pSB1K3.His⋅yCCS 2: pK H⋅y2 25/8

Sequencing

15 μl DNA + 1.5 μl 10 μM pSB-VF2

  • pK.H.S1: ASB0045 979
    • pSB1K3.His⋅SOD 1
  • pK.H.S3: ASB0045 980
    • pSB1K3.His⋅SOD 3
  • pK.H.y1: ASB0045 981
    • pSB1K3.His⋅yCCS 1
  • pK.H.y2: ASB0045 982
    • pSB1K3.His⋅yCCS 2

Transfer of RFP cassette into pEX

Transformation results

From 24/8

Good colony yield but no red. Recalled that the RFP cassette is expressed from a LacI-sensitive promoter; since pEX expresses the LacI repressor, IPTG has to be added to induce expression of the RFP cassette.

Transformation

Plated an Amp 100 plate with 50 μl 0.1 M IPTG. LB agar was then plated with quick-transformed cells, transformed with the 24/8 ligation mix.

  • 1.5 μl ligation mix

Johan

  • Ligate cut bFGF into cut C-vector

1 µl vector

5 µl bFGF

2 µl 10x buffer

1 µl T4 ligase

11 µl H2O

20 min 22 °C

Then heat-inactivation

  • Transformation of bFGF+C





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