Team:Stockholm/25 August 2010

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==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/&mu;l
 +
!A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pSB1K3.His&sdot;SOD 1
 +
|align="center"|52.52
 +
|align="center"|1.73
 +
|align="center"|127.1
 +
|align="center"|1.84
 +
|-
 +
|pSB1K3.His&sdot;SOD 3
 +
|align="center"|41.13
 +
|align="center"|1.74
 +
|align="center"|101.8
 +
|align="center"|1.84
 +
|-
 +
|pSB1K3.His&sdot;yCCS 1
 +
|align="center"|67.48
 +
|align="center"|1.79
 +
|align="center"|133.2
 +
|align="center"|1.86
 +
|-
 +
|pSB1K3.His&sdot;yCCS 2
 +
|align="center"|37.63
 +
|align="center"|1.87
 +
|align="center"|146.7
 +
|align="center"|1.88
 +
|}
 +
 +
New ON cultures of pSB1K3.His&sdot;SOD 1 and pSB1K3.His&sdot;yCCS 2 were set for plasmid prep.
 +
*5 ml LB + Cm 25; 37 &deg;C, 250 rpm.
 +
 +
====Glycerol stocks====
 +
*'''pMA.His:''' pMA.His 25/8
 +
*'''pSB1K3.yCCS&sdot;His 2:''' pK y&sdot;H2 25/8
 +
*'''pSB1K3.yCCS&sdot;His 3:''' pK y&sdot;H3 25/8
 +
*'''pSB1K3.His&sdot;SOD 1:''' pK H&sdot;S1 25/8
 +
*'''pSB1K3.His&sdot;SOD 3:''' pK H&sdot;S3 25/8
 +
*'''pSB1K3.His&sdot;yCCS 1:''' pK H&sdot;y1 25/8
 +
*'''pSB1K3.His&sdot;yCCS 2:''' pK H&sdot;y2 25/8
 +
 +
====Sequencing====
 +
15 &mu;l DNA + 1.5 &mu;l 10 &mu;M pSB-VF2
 +
*'''pK.H.S1:''' ASB0045 979
 +
**pSB1K3.His&sdot;SOD 1
 +
*'''pK.H.S3:''' ASB0045 980
 +
**pSB1K3.His&sdot;SOD 3
 +
*'''pK.H.y1:''' ASB0045 981
 +
**pSB1K3.His&sdot;yCCS 1
 +
*'''pK.H.y2:''' ASB0045 982
 +
**pSB1K3.His&sdot;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 &mu;l 0.1 M IPTG. LB agar was then plated with quick-transformed cells, transformed with the 24/8 ligation mix.
 +
*1.5 &mu;l 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
 +
{{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





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

Retrieved from "http://2010.igem.org/Team:Stockholm/25_August_2010"