Mouse tissue lysis for genotyping

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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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==== tissue lysis to release DNA ====
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* per tail or tissue chunk (tissue degradation is sped up if tissue/tail is cut into smaller bits):
* per tail or tissue chunk (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
** 100 µl Taq DNA polymerase 10x Reaction buffer with MgCl2 (e.g. Promega #M1883) diluted 1:10 in ddH2O
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* incubate for 3h to o/n at 56°C at 300 rpm
* incubate for 3h to o/n at 56°C at 300 rpm
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proteinase K inactivation:
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==== proteinase K inactivation ====
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* 95°C for 20 min at 300 rpm
* 95°C for 20 min at 300 rpm
* centrifuge at 13000 rpm for 3 min and transfer supernatant (80 µl) into new eppi
* 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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==== storage before PCR ====
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* 4°C short term, -20°C long term
* 4°C short term, -20°C long term

Revision as of 09:27, 18 April 2007

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.

tissue lysis to release DNA

  • per tail or tissue chunk (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 56°C at 300 rpm

proteinase K inactivation

  • 95°C for 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
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