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-	[[Melbourne/Protocols|<Back to protocols>]]	+	[[Melbourne/Protocols for Standard Methods |<Return to list of protocols>]] [[Melbourne|  <Team home page>]]
		+	*Applications:
		+	*#Identification of insert presence
-	===For 20uL reation volume===	+	*Time to complete protocol:
-	====Reaction Mixture====	+	**Lab time: 15min, 15min.
-	*0.5uL Enzyme 1	+	**Waiting time: 1-3hours
-	*0.5uL Enzyme 2	+	*Approximate cost of materials: $0.00
-	**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.	+
		+	====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

• Restriction enzymes and buffer
• BSA
• DNA for digestion
• milliQ water
• Loading dye for DNA gel

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

• microcentrifuge: 1.7ml
• Ice box and ice
• 37 degree incubator
• Pipettes and Tips

References