Team:Groningen/Notebook/17 September 2009

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(Difference between revisions)
(New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== '''Planning''' :→ {{todo}} work out the wiki page for GVP :→ {{done}} make a doodle for presentation planning (1-19...)
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|Yes (MvaI/XhoI)
|Yes (MvaI/XhoI)
|}
|}
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'''Restriction for Control and Assembly'''
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The plasmids from the o.n. precultures of pArsR-GVP and pNL29 were cut with PstI/EcoRI and MvaI/XhoI to cut out the entire part between the pre- and suffix and wanted fragment.
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 +
{|cellpadding="2" cellspacing="1" border="4"
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|'''Plasmid'''
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|'''Amount μL'''
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|'''MQ μL'''
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|'''Fast digest buffer'''
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|'''EcoRI fast digest enzyme'''
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|'''PstI fast digest enzyme'''
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|-
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|pArsR-GVP no.1
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|16.0
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|x
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|3.0
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|1.0
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|1.0
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|-
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|pArsR-GVP no.2
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|16.0
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|x
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|3.0
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|1.0
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|1.0
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|-
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|pArsR-GVP no.2
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|16.0
 +
|x
 +
|3.0
 +
|1.0
 +
|1.0
 +
|-
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|'''Plasmid'''
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|'''Amount μL'''
 +
|'''MQ μL'''
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|'''R-digest buffer'''
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|'''MvaI digest enzyme'''
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|'''XhoI digest enzyme'''
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|-
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|pNL29
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|16.0
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|x
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|3.0
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|1.0
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|1.0
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|}
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 +
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[[Image:17-9 no.1.jpg|340px]] [[Image:Generulers_1kb_marker_Fermentas.jpg]]
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:→ From left to right: 1kB ladder, pNL29 (orange dye), pNL29 (blue dye), pArsR-GVP no.1, pArsR-GVP no.2, pArsR-GVP no.3
===Transporters===
===Transporters===

Latest revision as of 09:00, 17 September 2009

Igemhomelogo.png

Wet

GVP Cluster

Planning

TODO work out the wiki page for GVP
DONE make a doodle for presentation planning (1-19 oct.)
TODO media attention
DONE place an ethics survey link on twitter


TODO clone pArsR-GVP into pSB2K3
TODO clone repeat out of GVP cluster
TODO make glycerol stocks of constructs
TODO enter info on part registry


Cultures

The overnight cultures with LB-amp100 medium of colonies E.coli TOP10 with pNL29 (3x), and pArsR-GVP (1-3) all showed expected growth of bacteria.


www.sigmaaldrich.com

Plasmid isolation

Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".

  • From each tube 4mL of culture (8mL for pNL29) was collected in a 2.0mL cup (tubes from pArsR-GVP, GVP and pNL29 were combined), and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
  • Plasmids were eluted with 30μL MQ and stored in the fridge


Concentrations

Plasmid Conc. ng/μL 260/280 260/230 -20 box (michael Restriction Control
pArsR-GVP (pSB2K3, no.1) 40.6 1.89 1.77 x Yes (EcoRI/PstI)
pArsR-GVP (pSB2K3, no.2) 47.3 1.87 2.01 x Yes (EcoRI/PstI)
pArsR-GVP (pSB2K3, no.3) 40.4 1.89 1.64 x Yes (EcoRI/PstI)
pNL29 26.4 2.10 1.64 x Yes (MvaI/XhoI)


Restriction for Control and Assembly

The plasmids from the o.n. precultures of pArsR-GVP and pNL29 were cut with PstI/EcoRI and MvaI/XhoI to cut out the entire part between the pre- and suffix and wanted fragment.

Plasmid Amount μL MQ μL Fast digest buffer EcoRI fast digest enzyme PstI fast digest enzyme
pArsR-GVP no.1 16.0 x 3.0 1.0 1.0
pArsR-GVP no.2 16.0 x 3.0 1.0 1.0
pArsR-GVP no.2 16.0 x 3.0 1.0 1.0
Plasmid Amount μL MQ μL R-digest buffer MvaI digest enzyme XhoI digest enzyme
pNL29 16.0 x 3.0 1.0 1.0


340px Generulers 1kb marker Fermentas.jpg

→ From left to right: 1kB ladder, pNL29 (orange dye), pNL29 (blue dye), pArsR-GVP no.1, pArsR-GVP no.2, pArsR-GVP no.3

Transporters

Metal Accumulation

Vectors

Dry

April
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
May
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 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
    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 31
August
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
31
September
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
October
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 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