Team:Groningen/Notebook/12 August 2009

From 2009.igem.org

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===GVP Cluster===
===GVP Cluster===
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{{todo}} isolate plasmids from o.n. cultures
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:→ {{done}} isolate plasmids from o.n. cultures
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:→ {{done}} run gel to check plasmid size (should be ~9000bp)
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:→ {{todo}} analyse results of gel
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{{todo}} run gel to check plasmid size (should be ~9000bp)
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[[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]]
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'''Plasmid Purification'''
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Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 medium] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 low] constitutive promoters and GVP with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit".
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* From each tube 4mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
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* Plasmids were eluted with 20μL MQ and stored in the fridge
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'''Gel'''
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From each cup with isolated plasmid 5μL was mixed with 1μL 6x loading buffer and incubated for 10 minutes. Together with 1kB ladder all samples were loaded on a 1% agarose gel with 4μL EtBr.
===Transporters===
===Transporters===

Revision as of 08:22, 12 August 2009

Igemhomelogo.png

Wet

GVP Cluster

DONE isolate plasmids from o.n. cultures
DONE run gel to check plasmid size (should be ~9000bp)
TODO analyse results of gel
www.sigmaaldrich.com

Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids pSB1AC3 with medium and low constitutive promoters and GVP with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".

  • From each tube 4mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
  • Plasmids were eluted with 20μL MQ and stored in the fridge

Gel

From each cup with isolated plasmid 5μL was mixed with 1μL 6x loading buffer and incubated for 10 minutes. Together with 1kB ladder all samples were loaded on a 1% agarose gel with 4μL EtBr.

Transporters

Metal Accumulation

MBP-ArsR fusion protein ligation was transformed for a second try, plated out on LB-amp100 (50 μL and concentrated after centrifugation) Positive control (pSB1AC3-high constitutive promotor) ∞ Negative control (pSB1AC3-ccdb)1 colony on the concentrated plate Ligations had no colonies.

Vectors

Dry

April
MTWTFSS
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6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May
MTWTFSS
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4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
MTWTFSS
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6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
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3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
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7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
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5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
November
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30