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 9: | 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 24: | 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 | ||
| Line 34: | Line 36: | ||
#Airdry pellet | #Airdry pellet | ||
#Resuspend pellet in MilliQ water | #Resuspend pellet in MilliQ water | ||
==BioCoder version== | |||
Following is the DNA extraction from tissue protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth|Vaishnavi]] | |||
====Text Output==== | |||
[[DNA extraction from tissue protocol]] | |||
====Source Code==== | |||
[[DNA extraction from tissue protocol - source code]] | |||
== See also == | |||
* [[Oneill Lab:DNA Extraction]] | |||
* [[Mouse tissue lysis for genotyping]] | |||
* [[DNA extraction - Salting Out]] | |||
== External links == | |||
* [[Image:3stars.png]] [http://www.riedlab.nci.nih.gov/publications/2439.2451_DNA_Prep_Fresh_Froz_Tiss.pdf DNA from fresh or frozen tissue by Ried lab] | |||
* [[Image:3stars.png]] [http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1178 DNA from liver by Bowtell and colleagues] - cached version from protocol online | |||
* [[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] | |||
[[Category:Protocol]] | |||
[[Category:In vivo]] | |||
[[Category:Mouse]] | |||
[[Category:DNA]] | |||
Latest revision as of 02:45, 19 November 2009
| 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
BioCoder version
Following is the DNA extraction from tissue protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi
Text Output
DNA extraction from tissue protocol
Source Code
DNA extraction from tissue protocol - source code
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
