Melbourne/Diagnostic Digest

From 2007.igem.org

< Melbourne(Difference between revisions)
 
(12 intermediate revisions not shown)
Line 1: Line 1:
-
===For 20uL reation volume===
+
[[Melbourne/Protocols for Standard Methods |<Return to list of protocols>]]  [[Melbourne|  <Team home page>]]
-
====Reaction Mixture====
+
*Applications:
-
*0.5uL Enzyme 1
+
*#Identification of insert presence
-
*0.5uL Enzyme 2
+
-
**Add enzymes last
+
-
*2uL appropriate 10x buffer (see table on fridge)
+
-
*2uL 10x BSA
+
-
*10uL MilliQ
+
-
*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.
+
 +
*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=====
 +
*[[Melbourne/primary Restriction enzymes|Restriction enzymes and buffer]]
 +
*[[Melbourne/primary BSA|BSA]]
 +
*DNA for digestion
 +
*[[Melbourne/primary milliq|milliQ water]]
 +
*[[Melbourne/primary dna loading|Loading dye for DNA gel]]
 +
 +
=====Method including controls=====
 +
======For 20uL reation volume======
 +
#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 [[Melbourne/primary milliq|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.
#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.
 +
 +
=====Equipement Required=====
 +
*[[Melbourne/1.7ml microcentrifuge|microcentrifuge: 1.7ml]]
 +
*Ice box and [[Melbourne/primary ice|ice]]
 +
*37 degree incubator
 +
*Pipettes and [[Melbourne/primary tips|Tips]]
 +
 +
=====References=====
 +
*
 +
 +
__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