Mouse tissue lysis for genotyping

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{{back to protocols}}
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This is a quick protocol for mouse tail and tissue lysis with proteinase K. It is commonly used to prepare templates for genotyping. Other protocols included detergents in the lysis buffer, but we found this simple protocol to work well with less hands-on time.
This is a quick protocol for mouse tail and tissue lysis with proteinase K. It is commonly used to prepare templates for genotyping. Other protocols included detergents in the lysis buffer, but we found this simple protocol to work well with less hands-on time.
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tissue lysis to release DNA:
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== material ==
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* per tail or tissue chunk (tissue degradation is sped up if tissue/tail is cut into smaller bits):
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* proteinase K
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** 100 µl Taq DNA polymerase 10x Reaction buffer with MgCl2 (e.g. Promega #M1883) diluted 1:10 in ddH2O
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* Taq 10x buffer
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** +2 µl proteinase K (20 mg/ml)
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* tabletop shaker/incubator
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* incubate for 3h to o/n at 56°C at 300 rpm
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== procedure ==
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=== tissue lysis to release DNA ===
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per tail or tissue chunk add (tissue degradation is sped up if tissue/tail is cut into smaller bits):
 +
* 100 µl Taq DNA polymerase 10x Reaction buffer with MgCl2 (e.g. Promega #M1883) diluted 1:10 in ddH2O
 +
* +2 µl proteinase K (20 mg/ml)
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* incubate for 3h to o/n at 50°C at 300 rpm
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:(proteinase K is stable over a broad pH range (4.0 to 12.5, optimum pH 8.0) and is also stable over the temperature range of 25 to 65°C)
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=== proteinase K inactivation ===
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* 95°C for 10-20 min at 300 rpm
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* centrifuge at 13000 rpm for 3 min and transfer supernatant (80 µl) into new eppi
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=== storage before PCR ===
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* 4°C short term, -20°C long term
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==BioCoder version==
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Following is the Mouse tissue lysis for genotyping 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]]
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====Text Output====
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[[Mouse tissue lysis for genotyping protocol]]
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====Source Code====
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[[Mouse tissue lysis for genotyping protocol - source code]]
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== see also ==
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* [[DNA extraction from tissue]]
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* [http://en.wikipedia.org/wiki/Genotyping Genotyping in Wikipedia]
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* [[PCR]] - how to do a PCR
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* [[Designing primers]] - how to design primers
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* [[PCR techniques]] - overview page
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== external links ==
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* [[Image:3stars.png]] [http://cancer.ucsd.edu/tgm/TailGenomicDNAPrep.pdf UCSD protocol genomic DNA from mouse tails]
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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
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* [http://www.protocol-online.org/prot/Molecular_Biology/DNA/DNA_Extraction___Purification/DNA_Extraction_from_Cell_and_Tissue/Tail_DNA_Extraction/ Protocol Online - tail DNA extraction protocols]
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[[Category:Protocol]]
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[[Category:In vivo]]
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[[Category:Mouse]]
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[[Category:DNA]]

Current revision

back to protocols

This is a quick protocol for mouse tail and tissue lysis with proteinase K. It is commonly used to prepare templates for genotyping. Other protocols included detergents in the lysis buffer, but we found this simple protocol to work well with less hands-on time.

Contents

material

  • proteinase K
  • Taq 10x buffer
  • tabletop shaker/incubator

procedure

tissue lysis to release DNA

per tail or tissue chunk add (tissue degradation is sped up if tissue/tail is cut into smaller bits):

  • 100 µl Taq DNA polymerase 10x Reaction buffer with MgCl2 (e.g. Promega #M1883) diluted 1:10 in ddH2O
  • +2 µl proteinase K (20 mg/ml)


  • incubate for 3h to o/n at 50°C at 300 rpm
(proteinase K is stable over a broad pH range (4.0 to 12.5, optimum pH 8.0) and is also stable over the temperature range of 25 to 65°C)

proteinase K inactivation

  • 95°C for 10-20 min at 300 rpm
  • centrifuge at 13000 rpm for 3 min and transfer supernatant (80 µl) into new eppi

storage before PCR

  • 4°C short term, -20°C long term


BioCoder version

Following is the Mouse tissue lysis for genotyping 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

Mouse tissue lysis for genotyping protocol

Source Code

Mouse tissue lysis for genotyping protocol - source code

see also

external links

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