Team:UNIPV-Pavia/Methods Materials/Digestion

From 2009.igem.org

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(→BioBrick digestion with restriction enzymes)
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'''Materials needed:'''
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*'''Roche restriction enzymes thawed on ice'''
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*'''Roche buffer H'''
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*'''Pre-warmed at 37°C bath'''
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*'''Cut and gel-extracted vector'''
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*'''ddH2O'''
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*To open vectors:
*To open vectors:
**a volume containing 1 µg of purified plasmid
**a volume containing 1 µg of purified plasmid
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**25 µl final volume
**25 µl final volume
**incubate at 37°C for 2 hours and 30 minutes
**incubate at 37°C for 2 hours and 30 minutes
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*'''Roche restriction enzymes'''
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*'''Roche buffer H'''
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*'''Pre-warmed at 37°C bath'''
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*'''Cut and gel-extracted vector'''
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*'''ddH2O'''
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Revision as of 11:47, 30 June 2009

EthanolPVanimation.gif



Protocols


BioBrick digestion with restriction enzymes

(estimated time: 3 hours)

  • To open vectors:
    • a volume containing 1 µg of purified plasmid
    • 2 µl of buffer H
    • 1 µl of first enzyme
    • 1 µl of second enzyme
    • 20 µl final volume
    • incubate at 37°C for 3 hours
  • To excide fragments:
    • 20 µl of purified plasmid when <7 µg have been extracted. A volume containing 7 µg otherwise.
    • 2.5 µl of buffer H
    • 1 µl of first enzyme
    • 1 µl of second enzyme
    • 25 µl final volume
    • incubate at 37°C for 2 hours and 30 minutes










  • Roche restriction enzymes
  • Roche buffer H
  • Pre-warmed at 37°C bath
  • Cut and gel-extracted vector
  • ddH2O




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