DNA extraction from tissue: Difference between revisions
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Protocol for extraction of DNA from tissue or embryos. | Protocol for extraction of DNA from tissue or embryos. | ||
== | == Proteinase K digestion == | ||
#Mix DNA extraction buffer | #Mix DNA extraction buffer | ||
#*98 μl [[Oneill_Lab:Chemicals#ReagentB|ReagentB]] | #*98 μl [[Oneill_Lab:Chemicals#ReagentB|ReagentB]] | ||
Line 11: | Line 11: | ||
#Incubate at 50°C overnight | #Incubate at 50°C overnight | ||
==PCI== | == Phenol/chloroform/isoamyl (PCI) == | ||
#Prepare PCI mix | #Prepare PCI mix | ||
##One part tris-saturated phenol to one part 24:1 Chloroform:Isoamyl alcohol | ##One part tris-saturated phenol to one part 24:1 Chloroform:Isoamyl alcohol | ||
Line 26: | Line 26: | ||
#Continue to precipitation | #Continue to precipitation | ||
==Ethanol | == Ethanol precipitation == | ||
#Add 2 volumes 100% EtOH | #Add 2 volumes 100% EtOH | ||
#Add 1/10 volume 3M Sodium Acetate pH 5.0 | #Add 1/10 volume 3M Sodium Acetate pH 5.0 | ||
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* [[Image:2stars.png]] [http://www.bio.com/protocolstools/protocol.jhtml?id=p1689 DNA extraction from tail or tissue] - at BioProtocol from unknown contributor | * [[Image:2stars.png]] [http://www.bio.com/protocolstools/protocol.jhtml?id=p1689 DNA extraction from tail or tissue] - at BioProtocol from unknown contributor | ||
* [http://www.protocol-online.org/prot/Molecular_Biology/DNA/DNA_Extraction___Purification/DNA_Extraction_from_Cell_and_Tissue/ Protocol Online links to DNA extraction protocols] | * [http://www.protocol-online.org/prot/Molecular_Biology/DNA/DNA_Extraction___Purification/DNA_Extraction_from_Cell_and_Tissue/ Protocol Online links to DNA extraction protocols] | ||
Revision as of 08:20, 16 January 2008
back to protocols | ||
Protocol for extraction of DNA from tissue or embryos.
Proteinase K digestion
- Mix DNA extraction buffer
- 98 μl ReagentB
- 2 μl ProteinaseK
- Mix fresh. 100 μl is enough for a small pea size chunk of tissue or one embryo
- Place small piece of tissue or embryo into a microfuge tube containing 100 μl of extraction buffer
- Incubate at 50°C overnight
Phenol/chloroform/isoamyl (PCI)
- Prepare PCI mix
- One part tris-saturated phenol to one part 24:1 Chloroform:Isoamyl alcohol
- Shake thoroughly to make emulsion
- Add one volume of PCI to extracted sample
- Shake tubes for 10 seconds
- Centrifuge at max speed for 5 minutes
- Remove aqueous phase to a new tube
- Repeat as needed
- Add one volume 24:1 chloroform:isoamyl alcohol
- Shake tubes for 10 seconds
- Centrifuge at max speed for 5 minutes
- Remove aqueous phase to a new tube
- Continue to precipitation
Ethanol precipitation
- Add 2 volumes 100% EtOH
- Add 1/10 volume 3M Sodium Acetate pH 5.0
- Centrifuge at max speed for 10 minutes
- Decant ethanol
- Add 150 μl 70% EtOH
- Centrifuge at max speed for 2 minutes
- Pipette out ethanol
- Airdry pellet
- Resuspend pellet in MilliQ water
See also
External links
- DNA from fresh or frozen tissue by Ried lab
- DNA from liver by Bowtell and colleagues - cached version from protocol online
- DNA extraction from tail or tissue - at BioProtocol from unknown contributor
- Protocol Online links to DNA extraction protocols