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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

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