Team:UNIPV-Pavia/Methods Materials/Transformation

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(New page: {{UNIPV-Pavia/Protocols}} <br> == '''Transformation''' == ''(estimated time: 3 hours and 30 min + 12-16 hours overnight incubation)'' <br> <br> '''Materials needed:''' *'''LB agar plates ...)
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== '''Transformation''' ==  
== '''Transformation''' ==  
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'''Materials needed:'''
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*'''LB agar plates with proper antibiotic added incubated at 37°C'''
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*Put 1-2 µl of DNA resuspension or ligation into thaw TOP10 cells tube (every tube contains approximately 60 µl of competent cells).
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*'''Thawed Invitrogen TOP10 cells (every tube contains approximately 60 µl of competent cells)'''
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*Incubate on ice for 30 min.
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*'''Resuspended DNA'''
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*'''SOC medium'''
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*Put 4-6 µl of DNA resuspension into TOP10 tube.
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*Incubate on ice for 30-45 min.
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*Heat shock: 42°C for 1 min.
*Heat shock: 42°C for 1 min.
*Put transformed TOP10 tube on ice and then add 250 µl SOC medium.
*Put transformed TOP10 tube on ice and then add 250 µl SOC medium.
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*Incubate 2 hours at 37°C, 220 rpm.
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*Incubate 1 hour at 37°C, 220 rpm.
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*Centrifuge 10 min, 1200 rpm.
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*Plate 200 µl of the solution on a proper agar plate.
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*Remove 150 ul of bacteria-free supernatant.
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*Plate the remaining part of solution (resuspending the bacteria) on a proper agar plate.
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*Incubate overnight at 37°C.
*Incubate overnight at 37°C.
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<tr bgcolor='yellow'><td>INGREDIENTS:
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'''LB agar plates with proper antibiotic'''<hr color='white' width='70%'>
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'''Invitrogen TOP10 cells'''<hr color='white' width='70%'>
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'''Resuspended DNA'''<hr color='white' width='70%'>
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'''SOC medium'''</font>
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Latest revision as of 10:26, 21 October 2009

EthanolPVanimation.gif



Protocols


Transformation

(estimated time: 3 hours and 30 min + 12-16 hours overnight incubation)

  • Put 1-2 µl of DNA resuspension or ligation into thaw TOP10 cells tube (every tube contains approximately 60 µl of competent cells).
  • Incubate on ice for 30 min.
  • Heat shock: 42°C for 1 min.
  • Put transformed TOP10 tube on ice and then add 250 µl SOC medium.
  • Incubate 1 hour at 37°C, 220 rpm.
  • Plate 200 µl of the solution on a proper agar plate.
  • Incubate overnight at 37°C.








INGREDIENTS:

LB agar plates with proper antibiotic
Invitrogen TOP10 cells
Resuspended DNA

SOC medium





Retrieved from "http://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Transformation"