Team:Groningen/Notebook/15 July 2009

From 2009.igem.org

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(Transporters)
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* Cells added to 3 ml Ty in a reaction tube
* Cells added to 3 ml Ty in a reaction tube
* Incubation 1 h in waterbath shaker 37°C
* Incubation 1 h in waterbath shaker 37°C
 +
 +
* The cells are plated on Ty agar (100 μg/ml)
 +
* On one plate 200 μL is streaked out. The rest is concentrated (centrifugation: 1 min, 14600rpm) in 200 μL and also plated.
 +
* Incubation o/n 37°C
===Transporters===
===Transporters===
-
We decided to make MasterMixes of our own. All but template, primers and taq.
+
We decided to make MasterMixes of our own. All but 1uL template, 2uL primers and 1uL taq for 25uL reactions.
{|
{|
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{| border="1"
{| border="1"
|+ '''1X MasterMix Buffer NH SO'''
|+ '''1X MasterMix Buffer NH SO'''
-
! Component  
+
! Component
 +
! concentrations
 +
! volumes
|-
|-
! dNTP  
! dNTP  
-
| 2.0 mM   
+
| 0.2 mM   
|8 uL
|8 uL
|-
|-
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|80 uL
|80 uL
|-
|-
-
!taq buffer (NH)SO
+
!taq buffer KCl
|1X
|1X
|100 uL
|100 uL
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|652 uL
|652 uL
|}
|}
-
 
-
 
|width="10"|
|width="10"|
|
|
<!--Tabel 2 hier-->
<!--Tabel 2 hier-->
 +
{| border="1"
{| border="1"
|+ '''1X MasterMix Buffer KCl'''
|+ '''1X MasterMix Buffer KCl'''
-
! Component  
+
! Component
 +
! concentrations
 +
! volumes
|-
|-
! dNTP  
! dNTP  
-
| 2.0 mM   
+
| 0.2 mM   
|8 uL
|8 uL
|-
|-
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|672 uL
|672 uL
|}
|}
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|width="10"|
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|
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<!--Tabel 3 hier-->
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|}
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|
|
<!--Tabel 3 hier-->
<!--Tabel 3 hier-->
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|}
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==Vectors==
 +
* overnight cultures were diluted 100, 200 or 400 x
 +
* 200 ul of culture per well
 +
* Measured for 16 h
 +
:: - every 15 min
 +
:: - @ 37&deg;  with shaking 6 mm
 +
:: - for mRFP1  excitation =590 nm
 +
::              excitation =610 nm
 +
 +
 +
 +
{| border="1"
 +
|+ '''96-wells Microtiter plate'''
 +
!
 +
! 1 LB
 +
! 2 LB
 +
! 3 LB
 +
! 4 LB
 +
! 5 LB
 +
! 6
 +
! 7 TY
 +
! 8 TY
 +
! 9 TY
 +
! 10 TY
 +
! 11 TY
 +
! 12
 +
|-
 +
! A 100x
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
|-
 +
! B 100x
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
|-
 +
! C 200x
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
|-
 +
! D 200x
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
|-
 +
! E 400x
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
|-
 +
! F 400x
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
| pSB1AC3 
 +
| pSB3K3
 +
| BBA_J23109
 +
| BBA_J23106
 +
| BBA_J23100
 +
|
 +
|-
 +
! G
 +
|
 +
|
 +
|
 +
|
 +
|
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|
 +
|
 +
|
 +
|
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|
 +
|
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|
 +
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|-
 +
! H
 +
|
 +
|
 +
|
 +
|
 +
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 +
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|
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|}
|}

Latest revision as of 12:45, 17 August 2009

[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]

Wet

GVP Cluster

Restriction for Assembly

The vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J61035 BBa_J61035]) containing [http://partsregistry.org/wiki/index.php/Part:BBa_I750016 GVP] cluster was cut with PstI and XbaI to create correct ends for insert in promotor vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002]).

  • 10μL MQ
  • 6μL plasmid in MQ (2μg) (329.7 ng/μL)
  • 2μL Fast digest buffer
  • 1μL PstI fast digest enzyme
  • 1μL XbaI fast digest enzyme

The vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002]) containing promotor [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] was cut with SpeI and PstI.

  • 4μL MQ
  • 12μL plasmid in MQ (155.4 ng/μL)
  • 2μL Fast digest buffer
  • 1μL SpeI fast digest enzyme
  • 1μL PstI fast digest enzyme

The cups were incubated in a heat block at 37°C for 30 min. followed by adding 4μL 6x loading buffer for gel electroforesis.


Gel electroforesis

24μL of each sample was loaded on a 1% agarose gel (devided over two slots) with EtBr and a 1kb ladder was used (see picture).

Gel 2009-07-15.jpg Generulers 1kb marker Fermentas.jpg

→ From left to right: Empty slot, 1kb Marker, Empty slot, HmtA PCR reaction, Empty slot, (2x) GVP, Empty slot, (2x) [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100], and Empty slot


Gel Purification of GVP

"High Pure PCR Product Purification Kit" from [http://www.roche-applied-science.com Roche] was used for gel purification.

  • ~200mg of gel containing the desired fragment was dissolved in 500μL binding buffer by heating to 60°C for 10 min. and carefully vortexing.
  • 250μL isopropanol was added to the tube and vortexed
  • dissolved gel was transfered to the column and centrifuged for 1 min.
  • column was washed with 500μL Wash Solution, centrifuged for 1 min., 200μL Wash solution was added and centrifuged for 1 min.
  • 15μL MQ was applied to the column, incubated for 3 min. at room temperature and centrifuged for 1 min. at full speed.


Concentration of GVP-vector

The concentration of isolated cut vector of GVP with EcoRI and XbaI was determined with the use of a nano-drop.

GVP-cluster eluted in MQ

  • 33.5 ng/μL
  • 1.65 (260/280)
  • 0.77 (260/230)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] + vector eluted in MQ

  • 14.2 ng/μL
  • 1.78 (260/280)
  • 1.01 (260/230)
→ Concentrations are just high enough for a ligation reaction to be performed, some additional tuning of the procedure is required!!


Ligation of [http://partsregistry.org/wiki/index.php/Part:BBa_I750016 GVP] into [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 vector]

Mix:

  • 1 μL T4 Ligase buffer
  • 1 μL Vector (J23100 restriction fragment, 14.2 ng/μL)
  • 7.6 μL Insert (GVP gene cluster, 33.5 ng/μL)
  • 0.5 μL T4 Ligase

Method:

  • The mixture is incubated 30 min, 26°C
  • To inactivate Ligase 10 min 70°C


Transformation of E.coli TOP10 cells with GVP + J23100

  • 2 μL Ligation mix added to cells from -80°C glycerol stock
  • 30 min incubation on ice
  • 5 min heat shock 37°C
  • 5 min on ice
  • Cells added to 3 ml Ty in a reaction tube
  • Incubation 1 h in waterbath shaker 37°C
  • The cells are plated on Ty agar (100 μg/ml)
  • On one plate 200 μL is streaked out. The rest is concentrated (centrifugation: 1 min, 14600rpm) in 200 μL and also plated.
  • Incubation o/n 37°C


Transporters

We decided to make MasterMixes of our own. All but 1uL template, 2uL primers and 1uL taq for 25uL reactions.

1X MasterMix Buffer NH SO
Component concentrations volumes
dNTP 0.2 mM 8 uL
MgCl 2.0 mM 80 uL
taq buffer KCl 1X 100 uL
MQ 652 uL
1X MasterMix Buffer KCl
Component concentrations volumes
dNTP 0.2 mM 8 uL
MgCl 1.5 mM 60 uL
taq buffer (NH)SO 1X 100 uL
MQ 672 uL


MasterMix3
Component Mg/K
MQ 28.5 uL
F mut1 2 uL
R mut2 2 uL
dNTP 2 uL
MgCl 3 uL
taq buffer KCl 5 uL
taq buffer (NH)SO 5 uL
taq polymerase 2.5 uL
PCR 3 program Temperature Time
Denaturing 95° 2.00 min
Start Cycles 25X
Denaturing 95° 30 sec
Annealing 55° 20 sec
Elongation 72° 2.10 min
End cycles
Final elongation 72° 10 min
Hold Forever

Vectors

  • overnight cultures were diluted 100, 200 or 400 x
  • 200 ul of culture per well
  • Measured for 16 h
- every 15 min
- @ 37° with shaking 6 mm
- for mRFP1 excitation =590 nm
excitation =610 nm


96-wells Microtiter plate
1 LB 2 LB 3 LB 4 LB 5 LB 6 7 TY 8 TY 9 TY 10 TY 11 TY 12
A 100x pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100 pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100
B 100x pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100 pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100
C 200x pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100 pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100
D 200x pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100 pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100
E 400x pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100 pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100
F 400x pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100 pSB1AC3 pSB3K3 BBA_J23109 BBA_J23106 BBA_J23100
G
H

Dry

April
MTWTFSS
    [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/1_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/2_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/3_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/4_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/5_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 5]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/6_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/7_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/8_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/9_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/10_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/11_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/12_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 12]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/13_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/14_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/15_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/16_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/17_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/18_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/19_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 19]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/20_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/21_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/22_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/23_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/24_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/25_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/26_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 26]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/27_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/28_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/29_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/30_April_2009&preload=Team:Groningen/Notebook/Day&action=edit 30]
May
MTWTFSS
        [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/1_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/2_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/3_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 3]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/4_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/5_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/6_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 6] [http://2009.igem.org/Team:Groningen/Notebook/7_May_2009 7] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/8_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/9_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/10_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 10]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/11_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/12_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/13_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/14_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/15_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/16_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/17_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 17]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/18_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/19_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/20_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/21_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/22_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/23_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/24_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 24]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/25_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/26_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/27_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 27] [http://2009.igem.org/Team:Groningen/Notebook/28_May_2009 28] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/29_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/30_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/31_May_2009&preload=Team:Groningen/Notebook/Day&action=edit 31]
June
MTWTFSS
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/1_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/2_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/3_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/4_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 4] [http://2009.igem.org/Team:Groningen/Notebook/5_June_2009 5] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/6_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/7_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 7]
[http://2009.igem.org/Team:Groningen/Notebook/8_June_2009 8] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/9_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/10_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/11_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/12_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/13_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/14_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 14]
[http://2009.igem.org/Team:Groningen/Notebook/15_June_2009 15] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/16_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 16] [http://2009.igem.org/Team:Groningen/Notebook/17_June_2009 17] [http://2009.igem.org/Team:Groningen/Notebook/18_June_2009 18] [http://2009.igem.org/Team:Groningen/Notebook/19_June_2009 19] [http://2009.igem.org/Team:Groningen/Notebook/20_June_2009 20] [http://2009.igem.org/Team:Groningen/Notebook/21_June_2009 21]
[http://2009.igem.org/Team:Groningen/Notebook/22_June_2009 22] [http://2009.igem.org/Team:Groningen/Notebook/23_June_2009 23] [http://2009.igem.org/Team:Groningen/Notebook/24_June_2009 24] [http://2009.igem.org/Team:Groningen/Notebook/25_June_2009 25] [http://2009.igem.org/Team:Groningen/Notebook/26_June_2009 26] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/27_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/28_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 28]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/29_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/30_June_2009&preload=Team:Groningen/Notebook/Day&action=edit 30]
July
MTWTFSS
    [http://2009.igem.org/Team:Groningen/Notebook/1_July_2009 1] [http://2009.igem.org/Team:Groningen/Notebook/2_July_2009 2] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/3_July_2009&preload=Team:Groningen/Notebook/Day&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/4_July_2009&preload=Team:Groningen/Notebook/Day&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/5_July_2009&preload=Team:Groningen/Notebook/Day&action=edit 5]
[http://2009.igem.org/Team:Groningen/Notebook/6_July_2009 6] [http://2009.igem.org/Team:Groningen/Notebook/7_July_2009 7] [http://2009.igem.org/Team:Groningen/Notebook/8_July_2009 8] [http://2009.igem.org/Team:Groningen/Notebook/9_July_2009 9] [http://2009.igem.org/Team:Groningen/Notebook/10_July_2009 10] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/11_July_2009&preload=Team:Groningen/Notebook/Day&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/12_July_2009&preload=Team:Groningen/Notebook/Day&action=edit 12]
[http://2009.igem.org/Team:Groningen/Notebook/13_July_2009 13] [http://2009.igem.org/Team:Groningen/Notebook/14_July_2009 14] [http://2009.igem.org/Team:Groningen/Notebook/15_July_2009 15] [http://2009.igem.org/Team:Groningen/Notebook/16_July_2009 16] [http://2009.igem.org/Team:Groningen/Notebook/17_July_2009 17] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/18_July_2009&preload=Team:Groningen/Notebook/Day&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/19_July_2009&preload=Team:Groningen/Notebook/Day&action=edit 19]
[http://2009.igem.org/Team:Groningen/Notebook/20_July_2009 20] [http://2009.igem.org/Team:Groningen/Notebook/21_July_2009 21] [http://2009.igem.org/Team:Groningen/Notebook/22_July_2009 22] [http://2009.igem.org/Team:Groningen/Notebook/23_July_2009 23] [http://2009.igem.org/Team:Groningen/Notebook/24_July_2009 24] [http://2009.igem.org/Team:Groningen/Notebook/25_July_2009 25] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/26_July_2009&preload=Team:Groningen/Notebook/Day&action=edit 26]
[http://2009.igem.org/Team:Groningen/Notebook/27_July_2009 27] [http://2009.igem.org/Team:Groningen/Notebook/28_July_2009 28] [http://2009.igem.org/Team:Groningen/Notebook/29_July_2009 29] [http://2009.igem.org/Team:Groningen/Notebook/30_July_2009 30] [http://2009.igem.org/Team:Groningen/Notebook/31_July_2009 31]
August
MTWTFSS
          [http://2009.igem.org/Team:Groningen/Notebook/1_August_2009 1] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/2_August_2009&preload=Team:Groningen/Notebook/Day&action=edit 2]
[http://2009.igem.org/Team:Groningen/Notebook/3_August_2009 3] [http://2009.igem.org/Team:Groningen/Notebook/4_August_2009 4] [http://2009.igem.org/Team:Groningen/Notebook/5_August_2009 5] [http://2009.igem.org/Team:Groningen/Notebook/6_August_2009 6] [http://2009.igem.org/Team:Groningen/Notebook/7_August_2009 7] [http://2009.igem.org/Team:Groningen/Notebook/8_August_2009 8] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/9_August_2009&preload=Team:Groningen/Notebook/Day&action=edit 9]
[http://2009.igem.org/Team:Groningen/Notebook/10_August_2009 10] [http://2009.igem.org/Team:Groningen/Notebook/11_August_2009 11] [http://2009.igem.org/Team:Groningen/Notebook/12_August_2009 12] [http://2009.igem.org/Team:Groningen/Notebook/13_August_2009 13] [http://2009.igem.org/Team:Groningen/Notebook/14_August_2009 14] [http://2009.igem.org/Team:Groningen/Notebook/15_August_2009 15] [http://2009.igem.org/Team:Groningen/Notebook/16_August_2009 16]
[http://2009.igem.org/Team:Groningen/Notebook/17_August_2009 17] [http://2009.igem.org/Team:Groningen/Notebook/18_August_2009 18] [http://2009.igem.org/Team:Groningen/Notebook/19_August_2009 19] [http://2009.igem.org/Team:Groningen/Notebook/20_August_2009 20] [http://2009.igem.org/Team:Groningen/Notebook/21_August_2009 21] [http://2009.igem.org/Team:Groningen/Notebook/22_August_2009 22] [http://2009.igem.org/Team:Groningen/Notebook/23_August_2009 23]
[http://2009.igem.org/Team:Groningen/Notebook/24_August_2009 24] [http://2009.igem.org/Team:Groningen/Notebook/25_August_2009 25] [http://2009.igem.org/Team:Groningen/Notebook/26_August_2009 26] [http://2009.igem.org/Team:Groningen/Notebook/27_August_2009 27] [http://2009.igem.org/Team:Groningen/Notebook/28_August_2009 28] [http://2009.igem.org/Team:Groningen/Notebook/29_August_2009 29] [http://2009.igem.org/Team:Groningen/Notebook/30_August_2009 30]
[http://2009.igem.org/Team:Groningen/Notebook/31_August_2009 31]
September
MTWTFSS
  [http://2009.igem.org/Team:Groningen/Notebook/1_September_2009 1] [http://2009.igem.org/Team:Groningen/Notebook/2_September_2009 2] [http://2009.igem.org/Team:Groningen/Notebook/3_September_2009 3] [http://2009.igem.org/Team:Groningen/Notebook/4_September_2009 4] [http://2009.igem.org/Team:Groningen/Notebook/5_September_2009 5] [http://2009.igem.org/Team:Groningen/Notebook/6_September_2009 6]
[http://2009.igem.org/Team:Groningen/Notebook/7_September_2009 7] [http://2009.igem.org/Team:Groningen/Notebook/8_September_2009 8] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/9_September_2009&preload=Team:Groningen/Notebook/Day&action=edit 9] [http://2009.igem.org/Team:Groningen/Notebook/10_September_2009 10] [http://2009.igem.org/Team:Groningen/Notebook/11_September_2009 11] [http://2009.igem.org/Team:Groningen/Notebook/12_September_2009 12] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/13_September_2009&preload=Team:Groningen/Notebook/Day&action=edit 13]
[http://2009.igem.org/Team:Groningen/Notebook/14_September_2009 14] [http://2009.igem.org/Team:Groningen/Notebook/15_September_2009 15] [http://2009.igem.org/Team:Groningen/Notebook/16_September_2009 16] [http://2009.igem.org/Team:Groningen/Notebook/17_September_2009 17] [http://2009.igem.org/Team:Groningen/Notebook/18_September_2009 18] [http://2009.igem.org/Team:Groningen/Notebook/19_September_2009 19] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/20_September_2009&preload=Team:Groningen/Notebook/Day&action=edit 20]
[http://2009.igem.org/Team:Groningen/Notebook/21_September_2009 21] [http://2009.igem.org/Team:Groningen/Notebook/22_September_2009 22] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/23_September_2009&preload=Team:Groningen/Notebook/Day&action=edit 23] [http://2009.igem.org/Team:Groningen/Notebook/24_September_2009 24] [http://2009.igem.org/Team:Groningen/Notebook/25_September_2009 25] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/26_September_2009&preload=Team:Groningen/Notebook/Day&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/27_September_2009&preload=Team:Groningen/Notebook/Day&action=edit 27]
[http://2009.igem.org/Team:Groningen/Notebook/28_September_2009 28] [http://2009.igem.org/Team:Groningen/Notebook/29_September_2009 29] [http://2009.igem.org/Team:Groningen/Notebook/30_September_2009 30]
October
MTWTFSS
      [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/1_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 1] [http://2009.igem.org/Team:Groningen/Notebook/2_October_2009 2] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/3_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/4_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 4]
[http://2009.igem.org/Team:Groningen/Notebook/5_October_2009 5] [http://2009.igem.org/Team:Groningen/Notebook/6_October_2009 6] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/7_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/8_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/9_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/10_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/11_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 11]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/12_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/13_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/14_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/15_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/16_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/17_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/18_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 18]
[http://2009.igem.org/Team:Groningen/Notebook/19_October_2009 19] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/20_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/21_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/22_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/23_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/24_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/25_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 25]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/26_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/27_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/28_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/29_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/30_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/31_October_2009&preload=Team:Groningen/Notebook/Day&action=edit 31]
November
MTWTFSS
            [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/1_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 1]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/2_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/3_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/4_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/5_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/6_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/7_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/8_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 8]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/9_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/10_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/11_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/12_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/13_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 13] [http://2009.igem.org/Team:Groningen/Notebook/14_November_2009 14] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/15_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 15]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/16_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 16] [http://2009.igem.org/Team:Groningen/Notebook/17_November_2009 17] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/18_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/19_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/20_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/21_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/22_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 22]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/23_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/24_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/25_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/26_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/27_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/28_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/29_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 29]
[http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/30_November_2009&preload=Team:Groningen/Notebook/Day&action=edit 30]