Team:Paris/August 18

From 2008.igem.org

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(→Miniprep and stock glycerol of L151)
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*[[Team:Paris/Notebook/Protocols#Minipreps (Kit_Qiagen)| Protocol Miniprep]]
*[[Team:Paris/Notebook/Protocols#Minipreps (Kit_Qiagen)| Protocol Miniprep]]
{|border="1" style="text-align: center"
{|border="1" style="text-align: center"
-
|'''Stock number'''
+
|'''Miniprep'''
|'''Biobricks'''
|'''Biobricks'''
|'''Description'''
|'''Description'''
|-
|-
|MP163.1
|MP163.1
-
|B0032
+
|rowspan="2"| B0032
 +
|rowspan="2"| RBS
|-
|-
|MP163.2
|MP163.2
-
|B0032
 
|-
|-
|MP164.1
|MP164.1
-
|E0422
+
|rowspan="2"| E0422
 +
|rowspan="2"| RBS+ ECFP+ LVA+ term
|-
|-
|MP164.2
|MP164.2
-
|E0422
 
|-
|-
|MP165.1
|MP165.1
-
|E0430
+
|rowspan="2"| E0430
 +
|rowspan="2"| RBS+ YFP+ LVA- term
|-
|-
|MP165.2
|MP165.2
-
|E0430
 
|-
|-
|MP166.1
|MP166.1
-
|E0432
+
|rowspan="2"| E0432
 +
|rowspan="2"| RBS+ YFP LVA+ term
|-
|-
|MP166.2
|MP166.2
-
|E0432
 
|-
|-
|MP167.1
|MP167.1
-
|E0420
+
|rowspan="2"| E0420
 +
|rowspan="2"| RBS+ ECFP LVA- term
|-
|-
|MP167.2
|MP167.2
-
|E0420
 
|-
|-
|MP168.1
|MP168.1
-
|I732078
+
|rowspan="2"| I732078
 +
|rowspan="2"| RBS+ mRFP LVA+ term
|-
|-
-
|MP168.2
+
|S168.2
-
|I732078
+
|}
|}

Revision as of 17:33, 20 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.


Miniprep and stock glycerol

Stock number Biobricks Description
S163.1 B0032 RBS
S163.2
S164.1 E0422 RBS+ ECFP+ LVA+ term
S164.2
S165.1 E0430 RBS+ YFP+ LVA- term
S165.2
S166.1 E0432 RBS+ YFP LVA+ term
S166.2
S167.1 E0420 RBS+ ECFP LVA- term
S167.2
S168.1 I732078 RBS+ mRFP LVA+ term
S168.2


Miniprep Biobricks Description
MP163.1 B0032 RBS
MP163.2
MP164.1 E0422 RBS+ ECFP+ LVA+ term
MP164.2
MP165.1 E0430 RBS+ YFP+ LVA- term
MP165.2
MP166.1 E0432 RBS+ YFP LVA+ term
MP166.2
MP167.1 E0420 RBS+ ECFP LVA- term
MP167.2
MP168.1 I732078 RBS+ mRFP LVA+ term
S168.2

Digestion of MP162

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 biobrocks 2008

Protocol

Biobrick Name Part Description
B0032 in pSB1A2 rbs (efficiency=0.3) Icon rbs.png
E0422 in pSB1A2 ECFP (LVA+) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
E0420 in pSB1A2 ECFP (LVA-) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
E0430 in pSB1A2 EYFP (LVA-) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
E0432 in pSB1A2 EYFP (LVA+) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
I732078 in pSB1A3 mRFP1 (LVA+) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
I13507 in pSB1A2 mRFP1 (LVA-) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
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