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From 2010.igem.org

Contents 1 Andreas 1.1 Transfer of MITF to pSB1C3 1.1.1 Transformation results 1.2 Construction of His⋅SOD fusions 1.2.1 Plasmid prep 1.2.2 Glycerol stocks 1.2.3 Sequencing 1.3 Transfer of RFP cassette into pEX 1.3.1 Transformation results 1.3.2 Transformation 2 Johan

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