Team:Stockholm/11 October 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
 +
==Nina==
 +
 +
===Continuation of protein purification===
 +
 +
I got help with the plugged drop column and got the fractions I needed of the lysis, wash and elution buffers.
 +
 +
These samples of 0.5 ml were stored in a freezer for running on an SDS gel.
 +
==Andreas==
==Andreas==
 +
===Removal of insertion in BioBrick suffixes===
 +
====Plasmid prep====
 +
''From 8/10 stored pellet''
 +
 +
*50 μl elution buffer.
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="3"|DNA concentration
 +
|-
 +
!Sample
 +
!width="60"|Conc [ng/μl]
 +
!width="60"|A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pSB1C3.SOD
 +
|align="center"|163.1
 +
|align="center"|1.94
 +
|}
 +
 +
====Digestion====
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|&nbsp;
 +
!width="50"|pSB1C3.<br />SOD
 +
|-
 +
|10X FastDigest buffer
 +
|align="center"|2
 +
|-
 +
|DNA (1 &mu;g)
 +
|align="center"|12.3
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|3.7
 +
|-
 +
|FD EcoRI
 +
|align="center"|1
 +
|-
 +
|FD SpeI
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
|}
 +
*Incubation: 37 &deg;C, 30 min
 +
 +
====Gel verification====
 +
[[image:Gelver_dig_pC.SOD_11oct.png|200px|thumb|right|'''Gel verification of pSB1C3.SOD digested with EcoRI and SpeI.'''<br />3 &mu;l &lambda;; 3 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder]]
 +
 +
1 % agarose, 140 V
 +
 +
'''Expected bands'''
 +
*Vector: 2175 bp
 +
*Insert: 495 bp
 +
 +
'''Results'''<br />
 +
Band slightly larger than expected, but this may also be due to bound restriction enzymes and/or other disturbances. Proceeded to gel extraction.
 +
 +
====Gel extraction====
 +
Digested pSB1C3.SOD separated on 1 % agarose gel at 110 V, 17 &mu;l sample volume. SOD band extracted and DNA, as well as DNA from samples stored in -20 &deg;C 7/10, were purified using the E.Z.N.A. Gel Extraction kit.
 +
 +
DNA measurements extremely low, but proceeded to ligation/cloning anyway.
 +
 +
====Ligation====
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|&nbsp;
 +
!pSB1C3.<br />IgGp
 +
!pSB1C3.<br />bGFG
 +
!pSB1C3.<br />ProtA
 +
!pSB1C3.<br />yCCS
 +
!pSB1C3.<br />SOD
 +
|-
 +
|10X T4 Ligase buffer
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|-
 +
|Vector DNA
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|3
 +
|-
 +
|Insert DNA
 +
|align="center"|14
 +
|align="center"|14
 +
|align="center"|14
 +
|align="center"|14
 +
|align="center"|14
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|-
 +
|T4 DNA ligase
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
|}
 +
*Incubation: 22 &deg;C, 15 min
 +
 +
====Transformation====
 +
*Mod. quick transformation
 +
**Top10
 +
**3 &mu;l ligation mix
 +
**30 min incubation on ice
 +
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== SOD activity assay ===
 +
 +
 +
*Nondenaturing gel electrophoresis
 +
**Staining with NBT
 +
 +
 +
*SOD activity test with enzyme producing O<sub>2</sub><sup>-</sup>
 +
**mix 750µl culture with 600µl ice cold ethanol:chloroform (5:3)
 +
**vortex 20s, incubate 3min, vortex again 20s
 +
**centrifuge at 2500g for 45min in 4&deg;C
 +
**keep supernatant on ice
 +
 +
{|
 +
! mix
 +
|
 +
|-
 +
| supernatant
 +
| rowspan="6" |
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*Measure A<sub>546</sub> for 10min at 25&deg;C
 +
 +
 +
 +
*xanthine oxidase activity ajusted to increase in A<sub>546</sub> 0.1 U/min
 +
|-
 +
| phosphate buffer 50mM
 +
|-
 +
| EDTA 5mM
 +
|-
 +
| bovine serum albumin 40mg/l
 +
|-
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| NBT 0.2g/l
 +
|-
 +
| xanthine + oxidase 0.1mM
 +
|}
 +
 +
 +
 +
 +
*SOD activity test with illumination
 +
 +
{|
 +
! mix
 +
|
 +
|
 +
|-
 +
| riboflavin
 +
| 1.17*10<sup>-6</sup>M
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| rowspan="8" |
 +
*Illuminate so the absorbance increases with 0.142U/6min
 +
 +
*A<sub>560</sub> linear increase with time
 +
|-
 +
| methionine
 +
| 0.01M
 +
|-
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| sodium cyanide
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| 2*10<sup>-5</sup>M
 +
|-
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| NBT
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| 5.6*10<sup>-5</sup>M
 +
|-
 +
| potassium phosphate
 +
| 0.05M
 +
|-
 +
| align="center" | pH=7.8
 +
|
 +
|-
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| SOD
 +
| 20-260ng/ml
 +
|-
 +
|
 +
| tot 3.0ml
 +
|}
 +
 +
=Johan=
 +
==Ligation==
 +
his-bFGF and bFGF-his into gel purified pEX, both insert and vector cut with XbaI and PstI
 +
 +
7 µl insert, 0,5 µl pEX
 +
 +
1 µl ligase T4
 +
 +
2 µl 10x buffer
 +
 +
9,5 µl H2O
 +
 +
1 h 37 °C
 +
 +
==Transformation==
 +
 +
3 µl of both pEX + his-bFGF and pEX + bFGF-his into top10 cells
 +
 +
==Overnight culture==
 +
Made overnight culture from 10 colonies of tat-bFGF-his which didnt seem to be cut last time
 +
 +
{{Stockholm/Footer}}

Latest revision as of 19:47, 27 October 2010

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

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Contents

Nina

Continuation of protein purification

I got help with the plugged drop column and got the fractions I needed of the lysis, wash and elution buffers.

These samples of 0.5 ml were stored in a freezer for running on an SDS gel.

Andreas

Removal of insertion in BioBrick suffixes

Plasmid prep

From 8/10 stored pellet

  • 50 μl elution buffer.
DNA concentration
Sample Conc [ng/μl] A260/A280
pSB1C3.SOD 163.1 1.94

Digestion

  pSB1C3.
SOD
10X FastDigest buffer 2
DNA (1 μg) 12.3
dH2O 3.7
FD EcoRI 1
FD SpeI 1
  20 μl
  • Incubation: 37 °C, 30 min

Gel verification

Gel verification of pSB1C3.SOD digested with EcoRI and SpeI.
3 μl λ; 3 μl sample.
λ = O'GeneRuler 1 kb DNA ladder

1 % agarose, 140 V

Expected bands

  • Vector: 2175 bp
  • Insert: 495 bp

Results
Band slightly larger than expected, but this may also be due to bound restriction enzymes and/or other disturbances. Proceeded to gel extraction.

Gel extraction

Digested pSB1C3.SOD separated on 1 % agarose gel at 110 V, 17 μl sample volume. SOD band extracted and DNA, as well as DNA from samples stored in -20 °C 7/10, were purified using the E.Z.N.A. Gel Extraction kit.

DNA measurements extremely low, but proceeded to ligation/cloning anyway.

Ligation

  pSB1C3.
IgGp 		pSB1C3.
bGFG 		pSB1C3.
ProtA 		pSB1C3.
yCCS 		pSB1C3.
SOD
10X T4 Ligase buffer 2 2 2 2 2
Vector DNA 3 3 3 3 3
Insert DNA 14 14 14 14 14
dH2O 0 0 0 0 0
T4 DNA ligase 1 1 1 1 1
  20 μl 20 μl 20 μl 20 μl 20 μl
  • Incubation: 22 °C, 15 min

Transformation

  • Mod. quick transformation
    • Top10
    • 3 μl ligation mix
    • 30 min incubation on ice



Mimmi

SOD activity assay

  • Nondenaturing gel electrophoresis
    • Staining with NBT


  • SOD activity test with enzyme producing O2-
    • mix 750µl culture with 600µl ice cold ethanol:chloroform (5:3)
    • vortex 20s, incubate 3min, vortex again 20s
    • centrifuge at 2500g for 45min in 4°C
    • keep supernatant on ice
mix
supernatant
  • Measure A546 for 10min at 25°C


  • xanthine oxidase activity ajusted to increase in A546 0.1 U/min
phosphate buffer 50mM
EDTA 5mM
bovine serum albumin 40mg/l
NBT 0.2g/l
xanthine + oxidase 0.1mM



  • SOD activity test with illumination
mix
riboflavin 1.17*10-6M
  • Illuminate so the absorbance increases with 0.142U/6min
  • A560 linear increase with time
methionine 0.01M
sodium cyanide 2*10-5M
NBT 5.6*10-5M
potassium phosphate 0.05M
pH=7.8
SOD 20-260ng/ml
tot 3.0ml

Johan

Ligation

his-bFGF and bFGF-his into gel purified pEX, both insert and vector cut with XbaI and PstI

7 µl insert, 0,5 µl pEX

1 µl ligase T4

2 µl 10x buffer

9,5 µl H2O

1 h 37 °C

Transformation

3 µl of both pEX + his-bFGF and pEX + bFGF-his into top10 cells

Overnight culture

Made overnight culture from 10 colonies of tat-bFGF-his which didnt seem to be cut last time





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

Retrieved from "http://2010.igem.org/Team:Stockholm/11_October_2010"