Team:Paris/September 3

From 2008.igem.org

(Difference between revisions)
(New page: {{Paris/Calendar_Links|September 2|September 4}} =Transformation of yesterday overnight (18 hours) ligation= Ligation '''L173''': *insert: '''D112''' (ES) rbs-TetR *vector: '''D187''' (E...)
(Transformation)
 
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{{Paris/Calendar_Links|September 2|September 4}}
{{Paris/Calendar_Links|September 2|September 4}}
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=Transformation of yesterday overnight (18 hours) ligation=
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=Construction of rbs-TetR-mRFP-LVA-tripart (L173)=
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[[Image:Part_icon_rbs.png]][[Image:icon_coding.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
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==Transformation ==
Ligation '''L173''':
Ligation '''L173''':
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<br>
<br>
'''Transformation of E. coli DH5 alpha competent cells'''
'''Transformation of E. coli DH5 alpha competent cells'''
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*100 µL of competent cells
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* Defroze of the cells on ice
-
*5 µL of DNA (ligation products or pUC19 for positive control)
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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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* 30 min on ice
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*heat shock 45 s at 42°C
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* Heat schock 45sec at 42°C
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*2 min on ice
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* 2 min on ice
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*0,9 mL SOC
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* Add 900 µL of SOC
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*1 hour at 37°C
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* Incubate 1h at 37°C
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*plating on LB + ampicilline
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* Centrifugate 5 min at 6000 rpm
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*overnight incubation at 37°C
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* Remove 800 µL
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* Plate the 200 µL left on LB + amplicilline
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* incubate O/N at 37°C

Latest revision as of 18:53, 16 September 2008

← Yesterday

↓ Calendar ↑

Tomorrow →

Construction of rbs-TetR-mRFP-LVA-tripart (L173)

Part icon rbs.pngIcon coding.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png

Transformation

Ligation L173:

  • insert: D112 (ES) rbs-TetR
  • vector: D187 (EX) mRFP-LVA-tripart

Insert:vector ratio : 3:1 or 4:1


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