Melbourne/Diagnostic Digest

From 2007.igem.org

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[[Melbourne/Protocols|<Back to protocols>]]
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[[Melbourne/Protocols for Standard Methods |<Return to list of protocols>]] [[Melbourne|  <Team home page>]]
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*Applications:
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*#Identification of insert presence
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===For 20uL reation volume===
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*Time to complete protocol:
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====Reaction Mixture====
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**Lab time: 15min, 15min.
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*0.5uL Enzyme 1
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**Waiting time: 1-3hours
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*0.5uL Enzyme 2
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*Approximate cost of materials: $0.00
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**Add enzymes last
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*2uL appropriate 10x buffer (see table on fridge)
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*2uL 10x BSA
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*10uL MilliQ
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*5uL DNA
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**If performing the same digest for multiple DNA samples make up mastermix of buffer enzyme soultion and make 15uL aliquots to which to add the DNA
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**Volume DNA may vary depending on concentration, vary water volume to achieve 20uL reaction volume.
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====Method from primary and secondary reagents====
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=====Primary & secondary Reagents Required including controls=====
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*[[Melbourne/primary Restriction enzymes|Restriction enzymes and buffer]]
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*[[Melbourne/primary BSA|BSA]]
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*DNA for digestion
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*[[Melbourne/primary milliq|milliQ water]]
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*[[Melbourne/primary dna loading|Loading dye for DNA gel]]
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=====Method including controls=====
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======For 20uL reation volume======
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#Make the following reaction mixture on ice.
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#*Reaction Mixture
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#**0.5uL Enzyme 1
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#**0.5uL Enzyme 2
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#***Add enzymes last only take out of the freezer once ready to add.
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#**2uL appropriate 10x buffer (see table on fridge)
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#**2uL 10x BSA
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#**10uL [[Melbourne/primary milliq|milliQ water]]
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#**5uL DNA
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#***If performing the same digest for multiple DNA samples make up mastermix of buffer enzyme soultion and make 15uL aliquots to which to add the DNA.
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#***Volume DNA may vary depending on concentration, vary water volume to achieve 20uL reaction volume.
#Incubate for 1-3 hrs at 37 degrees.
#Incubate for 1-3 hrs at 37 degrees.
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#Stop reaction with addition of 5uL 6x DNA loading dye.
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#Stop reaction with addition of 5uL 6x [[Melbourne/primary dna loading| DNA Loading dye]].
#Can be stored at -20 or run on gel immediately.
#Can be stored at -20 or run on gel immediately.
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=====Equipement Required=====
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*[[Melbourne/1.7ml microcentrifuge|microcentrifuge: 1.7ml]]
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*Ice box and [[Melbourne/primary ice|ice]]
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*37 degree incubator
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*Pipettes and [[Melbourne/primary tips|Tips]]
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=====References=====
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*
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__NOTOC__

Latest revision as of 11:13, 29 September 2007

<Return to list of protocols> <Team home page>

  • Applications:
    1. Identification of insert presence
  • Time to complete protocol:
    • Lab time: 15min, 15min.
    • Waiting time: 1-3hours
  • Approximate cost of materials: $0.00

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls
Method including controls
For 20uL reation volume
  1. Make the following reaction mixture on ice.
    • Reaction Mixture
      • 0.5uL Enzyme 1
      • 0.5uL Enzyme 2
        • Add enzymes last only take out of the freezer once ready to add.
      • 2uL appropriate 10x buffer (see table on fridge)
      • 2uL 10x BSA
      • 10uL milliQ water
      • 5uL DNA
        • If performing the same digest for multiple DNA samples make up mastermix of buffer enzyme soultion and make 15uL aliquots to which to add the DNA.
        • Volume DNA may vary depending on concentration, vary water volume to achieve 20uL reaction volume.
  2. Incubate for 1-3 hrs at 37 degrees.
  3. Stop reaction with addition of 5uL 6x DNA Loading dye.
  4. Can be stored at -20 or run on gel immediately.
Equipement Required
References