Team:UNIPV-Pavia/Protocols/Digestion

From 2008.igem.org

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*[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]]
*[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]]
*[[Team:UNIPV-Pavia/Protocols/GelExtraction|DNA gel extraction]]
*[[Team:UNIPV-Pavia/Protocols/GelExtraction|DNA gel extraction]]
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*[[Team:UNIPV-Pavia/Protocols/Precipitation|DNA precipitation with sodium acetate]]
*[[Team:UNIPV-Pavia/Protocols/AntarcticPhosphatase|Antarctic Phosphatase]]
*[[Team:UNIPV-Pavia/Protocols/AntarcticPhosphatase|Antarctic Phosphatase]]
*[[Team:UNIPV-Pavia/Protocols/Ligation|Ligation]]
*[[Team:UNIPV-Pavia/Protocols/Ligation|Ligation]]
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*'''ddH2O'''
*'''ddH2O'''
<br>
<br>
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*
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*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
<br>
<br>

Latest revision as of 12:52, 1 July 2008

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The protocols we used


BioBrick digestion with restriction enzymes

(estimated time: 3 hours)

Materials needed:

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


  • 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