Team:Paris/September 5

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(→Purification of D112 (rbs-TetR))
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{{Paris/Calendar_Links|September 4|September 6}}
{{Paris/Calendar_Links|September 4|September 6}}
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=Purification of D112 (rbs-TetR)=
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=Purification of yesterday digestion=
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==Electrophoresis and gel extraction==
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[[Image:KR000300.JPG|thumb|D112 before gel excision]]
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{|border="1" style="text-align: center"
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|'''Digestion n°'''
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|'''DNA substrat'''
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|'''Digestion by'''
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|-
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|D112
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|MP106: S03879 (rbs-TetR) in pSB1A2
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|EcoRI & SpeI
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|-
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|}
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[[Image:KR000300.JPG|thumb|D112 before extraction]]
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==Electrophoresis and gel extraction==
1% agarose gel
1% agarose gel
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Qiaquick Gel Extraction Kit
Qiaquick Gel Extraction Kit
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*elution in 30 µL of EB buffer
==Concentration measurement==
==Concentration measurement==
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|-
|-
|}
|}
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=Constructions=
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[[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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==List of the constructions used to transformate==
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{|border="1" style="text-align: center"
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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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|-
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|L173
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|D112
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|D187
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|rbs-tetR-mRFP-LVA+
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|-
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|L183
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|D134
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|D187
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| pFlhB - mRFP LVA+
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|-
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|L184
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|D149
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|D198
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| pFliL - ECFP LVA+
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|-
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|L185
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|D149
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|D199
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| pFliL - ECFP LVA-
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|}
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 +
==Transformation of E. coli DH5 alpha competent cells==
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* Defroze of the cells on ice
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* Add 5 µL of DNA in 100 µL of competent cells (ligation products or pUC19 for positive control)
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* 30 min on ice
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* Heat schock 45sec at 42°C
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* 2 min on ice
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* Add 900 µL of SOC
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* Incubate 1h at 37°C
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* Centrifugate 5 min at 6000 rpm
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* 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

← Yesterday

↓ Calendar ↑

Tomorrow →

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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