Team:Stockholm/12 October 2010

From 2010.igem.org

(Difference between revisions)
(New page: {{Stockholm/Top2}} ==Andreas==)
 
(5 intermediate revisions not shown)
Line 1: Line 1:
{{Stockholm/Top2}}
{{Stockholm/Top2}}
 +
==Nina==
 +
 +
===Gel clean up===
 +
 +
I loaded samples in an agarose gel 1 % and ran at 80 V.
 +
 +
Samples:
 +
 +
*Protein A_TAT_N, _Tra10_N and _LMWP_N
 +
*Fusion_CPP1, TAT_C ans _CPP3
 +
*Fusion_TAT_N, _Tra10_N and _LMWP_N
 +
 +
The procedure was according to the description in protocols.
 +
 +
Weight of all samples: 200 mg
 +
 +
200 * 3 = 600 ul QXI
 +
 +
In step 3 I added 10 ul of QIAEXII to each sample. 
 +
 +
===Polyacrylamide gel===
 +
 +
I ran a gel of the purified SOD.His (N terminal) to check if there had been any purification of the protein.
 +
 +
Ladder: PageRuler Unst. Protein Ladder
 +
 +
Arrangement on the gel:
 +
 +
[[Image:Aq18.jpg]]
 +
 +
Gel:
 +
 +
[[Image:Aq19.jpg|300px]]
 +
 +
The gel was incubated in coomassie blue staining and destained on shake.
 +
 +
Unfortunately there were no bands representing SOD.His (~17 kDa) on the gel, which must mean that there was too small culture (12 ml) and that the cells have not been lysed properly. I will redo this work but with more start culture and lyse the cells in a more effiecient way.
 +
 +
 +
The polyacrylamide gel was prepared by the SDS-PAGE mixtures described in protocols.
 +
==Andreas==
==Andreas==
 +
===Removal of insertion in BioBrick suffixes===
 +
====Transformation results====
 +
Good colony yield on all plates. A few red colonies discovered on all plates, indicating that some partly digested pSB1C3 vector, still containing the RFP insert, had been excised during gel extraction.
 +
 +
====Colony PCR====
 +
''From 11/10 transformations'''
 +
 +
#pSB1C3.IgGp A-C
 +
#pSB1C3.bFGF A-C
 +
#pSB1C3.ProtA A-C
 +
#pSB1C3.yCCS A-C
 +
#pSB1C3.SOD A-C
 +
 +
*Standard colony PCR settings
 +
**1:15 elongation time
 +
 +
====Gel verification====
 +
[[image:ColPCR_reminserts_gel1_12oct.png|200px|thumb|right|'''Gel 1. Colony PCR gel verification of cloned with insertion removed.'''<br />3 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
 +
[[image:ColPCR_reminserts_gel2_12oct.png|200px|thumb|right|'''Gel 2. Colony PCR gel verification of cloned with insertion removed.'''<br />3 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = O'GeneRuler 50 bp DNA ladder.]]
 +
'''Gel 1:''' 1 % agarose, 140 V<br />
 +
'''Gel 2:''' 1 % agarose, 160 V
 +
 +
'''Expected bands'''
 +
#pSB1C3.IgGp: 1262 bp
 +
#pSB1C3.bFGF: 794 bp
 +
#pSB1C3.ProtA: 506 bp
 +
#pSB1C3.yCCS: 1076 bp
 +
#pSB1C3.SOD: 791 bp
 +
 +
'''Results'''
 +
#All clones correct.
 +
#All clones correct.
 +
#All clones correct.
 +
#Correct size for clone C, weak band for clone B. Clone A probably carrying an RFP insert.
 +
#All clones correct.
 +
 +
====ON cultures====
 +
*5 ml LB + Cm 25; 37 &deg;C, 225 rpm
 +
**1C, 2C, 3C, 4C, 5C
 +
 +
===BL21 transformation and growth test===
 +
Testing BL21 cells with/without CPP expression by transforming and inducing with IPTG.
 +
 +
*50 &mu;l 0.1 M IPTG
 +
*40 &mu;l competent cells
 +
**pEX.SOD&sdot;His
 +
**pEX.nTra10&sdot;SOD&sdot;His
 +
**pEX.nTAT&sdot;SOD&sdot;His
 +
**pEX.nLMWP&sdot;SOD&sdot;His
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== SOD activity assay ===
 +
 +
*Kits?
 +
**Biosite/trevigen  6000sek
 +
**Biovision/labinova 3000sek, ca 1week delivery time
 +
**''Cayman''
 +
**''Cellbiolabs''
 +
**''Merch''
 +
**Abcam 3600sek
 +
**Probior
 +
**Sigma-Aldrich 3000sek
 +
**''T-cell technology inc.''
 +
 +
 +
 +
*Kit from Sigma-Aldrich
 +
**sponsored with a very good offer
 +
 +
{{Stockholm/Footer}}

Latest revision as of 11:09, 26 October 2010

Home Project Idea Lab Work Results Modelling Team       Follow us on and        2010.igem.org

↳   Notebook Safety Protocols


Contents

Nina

Gel clean up

I loaded samples in an agarose gel 1 % and ran at 80 V.

Samples:

  • Protein A_TAT_N, _Tra10_N and _LMWP_N
  • Fusion_CPP1, TAT_C ans _CPP3
  • Fusion_TAT_N, _Tra10_N and _LMWP_N

The procedure was according to the description in protocols.

Weight of all samples: 200 mg

200 * 3 = 600 ul QXI

In step 3 I added 10 ul of QIAEXII to each sample.

Polyacrylamide gel

I ran a gel of the purified SOD.His (N terminal) to check if there had been any purification of the protein.

Ladder: PageRuler Unst. Protein Ladder

Arrangement on the gel:

Aq18.jpg

Gel:

Aq19.jpg

The gel was incubated in coomassie blue staining and destained on shake.

Unfortunately there were no bands representing SOD.His (~17 kDa) on the gel, which must mean that there was too small culture (12 ml) and that the cells have not been lysed properly. I will redo this work but with more start culture and lyse the cells in a more effiecient way.


The polyacrylamide gel was prepared by the SDS-PAGE mixtures described in protocols.

Andreas

Removal of insertion in BioBrick suffixes

Transformation results

Good colony yield on all plates. A few red colonies discovered on all plates, indicating that some partly digested pSB1C3 vector, still containing the RFP insert, had been excised during gel extraction.

Colony PCR

From 11/10 transformations'

  1. pSB1C3.IgGp A-C
  2. pSB1C3.bFGF A-C
  3. pSB1C3.ProtA A-C
  4. pSB1C3.yCCS A-C
  5. pSB1C3.SOD A-C
  • Standard colony PCR settings
    • 1:15 elongation time

Gel verification

Gel 1. Colony PCR gel verification of cloned with insertion removed.
3 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.
Gel 2. Colony PCR gel verification of cloned with insertion removed.
3 μl λ; 5 μl sample.
λ = O'GeneRuler 50 bp DNA ladder.

Gel 1: 1 % agarose, 140 V
Gel 2: 1 % agarose, 160 V

Expected bands

  1. pSB1C3.IgGp: 1262 bp
  2. pSB1C3.bFGF: 794 bp
  3. pSB1C3.ProtA: 506 bp
  4. pSB1C3.yCCS: 1076 bp
  5. pSB1C3.SOD: 791 bp

Results

  1. All clones correct.
  2. All clones correct.
  3. All clones correct.
  4. Correct size for clone C, weak band for clone B. Clone A probably carrying an RFP insert.
  5. All clones correct.

ON cultures

  • 5 ml LB + Cm 25; 37 °C, 225 rpm
    • 1C, 2C, 3C, 4C, 5C

BL21 transformation and growth test

Testing BL21 cells with/without CPP expression by transforming and inducing with IPTG.

  • 50 μl 0.1 M IPTG
  • 40 μl competent cells
    • pEX.SOD⋅His
    • pEX.nTra10⋅SOD⋅His
    • pEX.nTAT⋅SOD⋅His
    • pEX.nLMWP⋅SOD⋅His


Mimmi

SOD activity assay

  • Kits?
    • Biosite/trevigen 6000sek
    • Biovision/labinova 3000sek, ca 1week delivery time
    • Cayman
    • Cellbiolabs
    • Merch
    • Abcam 3600sek
    • Probior
    • Sigma-Aldrich 3000sek
    • T-cell technology inc.


  • Kit from Sigma-Aldrich
    • sponsored with a very good offer





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

Retrieved from "http://2010.igem.org/Team:Stockholm/12_October_2010"