Team:Paris/September 5

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=Construction of pFlhB-mRFP-LVA-tripart & pFliL-ECFP-LVA-tripart=
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=Constructions=
[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
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*L183 = pFlhB - mRFP LVA+
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==List of the constructions used to transformate==
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*L184 = pFliL - ECFP LVA+
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{|border="1" style="text-align: center"
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*L185 = pFliL - ECFP LVA-
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|'''Ligation n°'''
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|'''Insert'''
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|'''Vector'''
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|'''Construction'''
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|-
 +
|L173
 +
|D112
 +
|D187
 +
|rbs-tetR-mRFP-LVA+
 +
|-
 +
|L183
 +
|D134
 +
|D187
 +
| pFlhB - mRFP LVA+
 +
|-
 +
|L184
 +
|D149
 +
|D198
 +
| pFliL - ECFP LVA+
 +
|-
 +
|L185
 +
|D149
 +
|D199
 +
| pFliL - ECFP LVA-
 +
|}
 +
 
 +
==Transformation of E. coli DH5 alpha competent cells==
 +
* Defroze of the cells on ice
 +
* Add 5 µL of DNA in 100 µL of competent cells (ligation products or pUC19 for positive control)
 +
* 30 min on ice
 +
* Heat schock 45sec at 42°C
 +
* 2 min on ice
 +
* Add 900 µL of SOC
 +
* Incubate 1h at 37°C
 +
* Centrifugate 5 min at 6000 rpm
 +
* Remove 800 µL
 +
* Plate the 200 µL left on LB + amplicilline
 +
* incubate O/N at 37°C

Latest revision as of 19:38, 16 September 2008

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Contents

Purification of yesterday digestion

D112 before gel excision
Digestion n° DNA substrat Digestion by
D112 MP106: S03879 (rbs-TetR) in pSB1A2 EcoRI & SpeI

Electrophoresis and gel extraction

1% agarose gel

Expected size Measured size
pSB1A2 (vector) 2079 bp 2 kb
rbs-TetR (insert) 703 bp 0,7 kb

Qiaquick Gel Extraction Kit

  • elution in 30 µL of EB buffer

Concentration measurement

Sample DNA concentration A260/A280
D112 8 µg/mL 2,31


Constructions

Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png

List of the constructions used to transformate

Ligation n° Insert Vector Construction
L173 D112 D187 rbs-tetR-mRFP-LVA+
L183 D134 D187 pFlhB - mRFP LVA+
L184 D149 D198 pFliL - ECFP LVA+
L185 D149 D199 pFliL - ECFP LVA-

Transformation of E. coli DH5 alpha competent cells

  • Defroze of the cells on ice
  • Add 5 µL of DNA in 100 µL of competent cells (ligation products or pUC19 for positive control)
  • 30 min on ice
  • Heat schock 45sec at 42°C
  • 2 min on ice
  • Add 900 µL of SOC
  • Incubate 1h at 37°C
  • Centrifugate 5 min at 6000 rpm
  • Remove 800 µL
  • Plate the 200 µL left on LB + amplicilline
  • incubate O/N at 37°C
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