Melbourne/Diagnostic Digest

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(→Equipement Required)
 
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=====Primary & secondary Reagents Required including controls=====
=====Primary & secondary Reagents Required including controls=====
*[[Melbourne/primary Restriction enzymes|Restriction enzymes and buffer]]
*[[Melbourne/primary Restriction enzymes|Restriction enzymes and buffer]]
-
*BSA
+
*[[Melbourne/primary BSA|BSA]]
*DNA for digestion
*DNA for digestion
*[[Melbourne/primary milliq|milliQ water]]
*[[Melbourne/primary milliq|milliQ water]]
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#**2uL appropriate 10x buffer (see table on fridge)
#**2uL appropriate 10x buffer (see table on fridge)
#**2uL 10x BSA
#**2uL 10x BSA
-
#**10uL MilliQ
+
#**10uL [[Melbourne/primary milliq|milliQ water]]
#**5uL DNA
#**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.
#***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.
#***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.
-
#Stop reaction with addition of 5uL 6x DNA loading dye.
+
#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.

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


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