Team:UNIPV-Pavia/Methods Materials/Digestion

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<h1>BioBrick digestion with restriction enzymes</h1>
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    <font face="Pristina" size="50" ><b>Protocols</b></font>
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=='''BioBrick digestion with restriction enzymes'''==
''(estimated time: 3 hours)''
''(estimated time: 3 hours)''
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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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<tr bgcolor='yellow'><td>INGREDIENTS:
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'''Roche restriction enzymes''' <hr color='white' width='70%'>
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'''Roche buffer H''' <hr color='white' width='70%'>
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'''Pre-warmed at 37°C bath''' <hr color='white' width='70%'>
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'''Cut and gel-extracted vector''' <hr color='white' width='70%'>
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'''ddH2O'''</font>
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Latest revision as of 10:29, 21 October 2009

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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






INGREDIENTS:

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

ddH2O





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