"
Team:Stockholm/25 August 2010
From 2010.igem.org
(Difference between revisions)
(New page: {{Stockholm/Top2}} ==Andreas==) |
(→Andreas) |
||
| Line 2: | Line 2: | ||
==Andreas== | ==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 | ||
| + | |||
| + | {|border="1" cellpadding="1" cellspacing="0" | ||
| + | !colspan="5"|DNA concentrations | ||
| + | |- | ||
| + | | | ||
| + | |colspan="2" align="center"|Original concentration | ||
| + | |colspan="2" align="center"|Conc. after evaporation | ||
| + | |- | ||
| + | !Sample | ||
| + | !ng/μl | ||
| + | !A<sub>260</sub>/A<sub>280</sub> | ||
| + | !ng/μl | ||
| + | !A<sub>260</sub>/A<sub>280</sub> | ||
| + | |- | ||
| + | |pSB1K3.His⋅SOD 1 | ||
| + | |align="center"|52.52 | ||
| + | |align="center"|1.73 | ||
| + | |align="center"|127.1 | ||
| + | |align="center"|1.84 | ||
| + | |- | ||
| + | |pSB1K3.His⋅SOD 3 | ||
| + | |align="center"|41.13 | ||
| + | |align="center"|1.74 | ||
| + | |align="center"|101.8 | ||
| + | |align="center"|1.84 | ||
| + | |- | ||
| + | |pSB1K3.His⋅yCCS 1 | ||
| + | |align="center"|67.48 | ||
| + | |align="center"|1.79 | ||
| + | |align="center"|133.2 | ||
| + | |align="center"|1.86 | ||
| + | |- | ||
| + | |pSB1K3.His⋅yCCS 2 | ||
| + | |align="center"|37.63 | ||
| + | |align="center"|1.87 | ||
| + | |align="center"|146.7 | ||
| + | |align="center"|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 | ||
Revision as of 22:37, 29 August 2010
| Home | Project Idea | Lab Work | Results | Modelling | Team | Follow us on  and 
| 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
