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Team:Stockholm/25 August 2010
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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. | 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 | *1.5 μl ligation mix | ||
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| + | ==Johan== | ||
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| + | * Ligate cut bFGF into cut C-vector | ||
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| + | 1 µl vector | ||
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| + | 5 µl bFGF | ||
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| + | 2 µl 10x buffer | ||
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| + | 1 µl T4 ligase | ||
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| + | 11 µl H2O | ||
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| + | 20 min 22 °C | ||
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| + | Then heat-inactivation | ||
| + | |||
| + | * Transformation of bFGF+C | ||
| + | {{Stockholm/Footer}} | ||
Latest revision as of 01:52, 28 October 2010
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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
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