Team:Paris/August 18

From 2008.igem.org

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=Construction of rbs-LasR-dble ter=
=Construction of rbs-LasR-dble ter=
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=='''Miniprep and stock glycerol of L151 (rbs-LasR - Double terminator)'''==
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==Miniprep and stock glycerol of L151 (rbs-LasR - Double terminator)==
*[[Team:Paris/Notebook/Protocols#Glycerol stocks| Protocol stocks]]
*[[Team:Paris/Notebook/Protocols#Glycerol stocks| Protocol stocks]]
*[[Team:Paris/Notebook/Protocols#Minipreps (Kit_Qiagen)| Protocol minprep]]
*[[Team:Paris/Notebook/Protocols#Minipreps (Kit_Qiagen)| Protocol minprep]]
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=Construction of : promotor-rbs-LasR - Dble ter=
=Construction of : promotor-rbs-LasR - Dble ter=

Revision as of 13:37, 30 August 2008

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Contents

Analysis of the cloning of OmpR* and EnvZ*

PCR screening

  • positive control: S158 (pSB3K3)
  • negative control: no template
  • screening PCR programm
  • elongation time: 1 min 30
  • primers: O18 & O19

Reaction mixture (25 µL total volume)

  • 12,5 µL Quick load PCR Mix
  • 0,5 µL O18
  • 0,5 µL O19
  • 12 µL water

We wanted to analyse the transformants of the ligations L133 (OmpR* in pSB1A2) and L134 (EnvZ* in pSB1A2) from the experience of august 9th/11th.
3 clones of L133 and 3 clones of L134 were analysed.
Each clone tested was then cultured in 7,5 mL LB + ampicilline.


Construction of rbs-LasR-dble ter

Miniprep and stock glycerol of L151 (rbs-LasR - Double terminator)

Miniprep number Stock number Ligation name Antibio Biobricks Description
MP162.1 S162.1 L151.1 Kan Part icon rbs.pngIcon coding.pngPart icon terminator.pngPart icon terminator.png rbs-LasR - Double terminator
MP162.2 S162.2 L151.2
MP162.3 S162.3 L151.3

Construction of : promotor-rbs-LasR - Dble ter

Digestion

Measurement of the concentration of MPs 162

Miniprep Name Concentration (µg/mL) Ratio 260/280
MP162.1 151 1.71
MP162.2 172 1.73
MP162.3 149 1.73

Digestion

Protocol

Digestion name Template DNA Enzymes Volume of DNA
D165 MP162.2 XbaI - PstI 5.81 µL

Gel Extraction

Gel Extraction of D165

Protocol


Well Sample Expected size Measured size
1 1kb ladder
2 MP162.2 2855 3000
3 no sample
4 D165.2 2057 / 798 2000 / 800
5 no sample
6 100pb ladder

Ligation

Protocol

Ligation Name Vector Name Volume Vector (µL) Insert Volume Insert (µL)
L153 D123.1 1.54 D165 1.69
L154 D103.2 1.14 D165 1.68
Control L153 D123.1 1.54 - -
Control L154 D103.2 1.14 - -

Culture of stable strains with biobricks 2008

Protocol

Biobrick Name Part Description
[http://partsregistry.org/Part:BBa_B0032 B0032] in [http://partsregistry.org/Part:pSB1A2 pSB1A2] rbs (efficiency=0.3) Icon rbs.png
[http://partsregistry.org/Part:BBa_E0422 E0422] in [http://partsregistry.org/Part:pSB1A2 pSB1A2] ECFP (LVA+) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
[http://partsregistry.org/Part:BBa_E0420 E0420] in [http://partsregistry.org/Part:pSB1A2 pSB1A2] ECFP (LVA-) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
[http://partsregistry.org/Part:BBa_E0430 E0430] in [http://partsregistry.org/Part:pSB1A2 pSB1A2] EYFP (LVA-) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
[http://partsregistry.org/Part:BBa_E0432 E0432] in [http://partsregistry.org/Part:pSB1A2 pSB1A2] EYFP (LVA+) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
[http://partsregistry.org/Part:BBa_I732078 I732078] in [http://partsregistry.org/Part:pSB1A3 pSB1A3] mRFP1 (LVA+) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
[http://partsregistry.org/Part:BBa_I13507 I13507] in [http://partsregistry.org/Part:pSB1A2 pSB1A2] mRFP1 (LVA-) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
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