Team:Stockholm/8 October 2010

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====Plasmid prep====
====Plasmid prep====
Spun down pSB1C3.SOD ON culture at 13,000 x ''g'', 10 min. Supernatant discarded and cell pellet stored in -20 °C for later plasmid prep.
Spun down pSB1C3.SOD ON culture at 13,000 x ''g'', 10 min. Supernatant discarded and cell pellet stored in -20 °C for later plasmid prep.
 +
 +
==Nina==
 +
 +
===Protein purification===
 +
 +
I performed a protein purification of SOD.His (N terminal). The pellet came from an IPTG induced 12 ml overnight culture.
 +
 +
Lysis buffer:
 +
 +
630 ul * 2 = 1260 ul lysis buffer
 +
 +
Wash buffer:
 +
 +
10X lysis buffer  12.6 ml
 +
 +
Elution buffer:
 +
 +
5X lysis buffer 6.3 ml
 +
 +
*PMSF
 +
 +
100 * volume = 1260 * 1
 +
 +
12.6 ul PMSF added in lysis buffer
 +
 +
*Imidazole
 +
 +
2 * volume = 1260 * 10*10^-3
 +
 +
6.3 ul imidazole added in lysis buffer
 +
 +
*Imidazole
 +
 +
2 * volume = 1260 * 20*10^-3
 +
 +
126 ul imidazole added in wash buffer
 +
 +
*Lysozyme
 +
 +
The tip of a spoon was added in lysis buffer
 +
 +
*DNase
 +
 +
20 ug/ml was added in lysis buffer
 +
 +
====column equilibration====
 +
 +
The work is carried out in a cold room.
 +
 +
*Ni-resin was vortexed and 1 ml was added in a drop column. 
 +
*5 ml wash buffer was added and run through the column (to equilibrate the Ni-resin).
 +
*The lysis mixed with the bacteria pellet was added in the column. However, the mixture seemed to plug the column and nothing could run through.
 +
 +
I left this in the cold room over the weekend to check with anyone at the department what to do and if I still can work with this material.
 +
 +
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== SOD activity assay ===
 +
 +
*Searching "web of science" for SOD activity assays
 +
 +
[[Image:SOD_activity.png‎|left|]]
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 +
 +
 +
 +
 +
 +
 +
 +
 +
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 +
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*Generates O<sub>2</sub><sup>-</sup>
 +
**Xanthine + xanthine oxidase
 +
**NADH + phenazine methosulphate
 +
**riboflavin + methionine
 +
 +
 +
*Reacts with O<sub>2</sub><sup>-</sup> and can be measured
 +
**cytochrome C
 +
**nitro-blue tetrazodium NBT
 +
**adrenaline
 +
**pyrogallol
 +
**hydroxylamine
 +
 +
 +
 +
*Catalase-like test?
 +
**No, since E. coli are catalase positive and SOD differences will not be detectable
 +
 +
 +
 +
*SOD activity test that chemically produces O<sub>2</sub><sup>-</sup>
 +
 +
{|
 +
! mix
 +
|
 +
|
 +
|-
 +
| graded levels of SOD
 +
| ?
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| rowspan="9" |
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*Incubate in RT, darkness for 10min
 +
 +
*Measure A<sub>540</sub>
 +
 +
*negative control <sup>= without NADH</sup>
 +
 +
|-
 +
| NBT
 +
| 1mg
 +
|-
 +
| phenazine methosulphate
 +
| 10µg
 +
|-
 +
| EDTA
 +
| 1µM
 +
|-
 +
| gelatin
 +
| 1mg
 +
|-
 +
| phosphate
 +
| 0.1M
 +
|-
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| NADH 1mM
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| 0.1ml
 +
|-
 +
| align="right" | tot
 +
| 3ml
 +
|-
 +
| pH=7.8
 +
|}
 +
 +
{{Stockholm/Footer}}

Latest revision as of 11:08, 26 October 2010

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

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Contents

Andreas

Birthday cake

Short day in lab - birthday celebrations!

Growth curve assay

Inoculated fresh LB with 0.3 mM IPTG with ON cultures to a startup OD of 0.2 and a total volume of 50 ml in 150 ml side-arm E-flasks.

Was advised to use a smaller cultures for the flask size. Experiment canceled after about an hour; will be repeated next week.

Removal of insertion in BioBrick suffixes

Plasmid prep

Spun down pSB1C3.SOD ON culture at 13,000 x g, 10 min. Supernatant discarded and cell pellet stored in -20 °C for later plasmid prep.

Nina

Protein purification

I performed a protein purification of SOD.His (N terminal). The pellet came from an IPTG induced 12 ml overnight culture.

Lysis buffer:

630 ul * 2 = 1260 ul lysis buffer

Wash buffer:

10X lysis buffer 12.6 ml

Elution buffer:

5X lysis buffer 6.3 ml

  • PMSF

100 * volume = 1260 * 1

12.6 ul PMSF added in lysis buffer

  • Imidazole

2 * volume = 1260 * 10*10^-3

6.3 ul imidazole added in lysis buffer

  • Imidazole

2 * volume = 1260 * 20*10^-3

126 ul imidazole added in wash buffer

  • Lysozyme

The tip of a spoon was added in lysis buffer

  • DNase

20 ug/ml was added in lysis buffer

column equilibration

The work is carried out in a cold room.

  • Ni-resin was vortexed and 1 ml was added in a drop column.
  • 5 ml wash buffer was added and run through the column (to equilibrate the Ni-resin).
  • The lysis mixed with the bacteria pellet was added in the column. However, the mixture seemed to plug the column and nothing could run through.

I left this in the cold room over the weekend to check with anyone at the department what to do and if I still can work with this material.



Mimmi

SOD activity assay

  • Searching "web of science" for SOD activity assays
SOD activity.png









  • Generates O2-
    • Xanthine + xanthine oxidase
    • NADH + phenazine methosulphate
    • riboflavin + methionine


  • Reacts with O2- and can be measured
    • cytochrome C
    • nitro-blue tetrazodium NBT
    • adrenaline
    • pyrogallol
    • hydroxylamine


  • Catalase-like test?
    • No, since E. coli are catalase positive and SOD differences will not be detectable


  • SOD activity test that chemically produces O2-
mix
graded levels of SOD  ?
  • Incubate in RT, darkness for 10min
  • Measure A540
  • negative control = without NADH
NBT 1mg
phenazine methosulphate 10µg
EDTA 1µM
gelatin 1mg
phosphate 0.1M
NADH 1mM 0.1ml
tot 3ml
pH=7.8





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

Retrieved from "http://2010.igem.org/Team:Stockholm/8_October_2010"