Mouse tissue lysis for genotyping protocol
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Solutions/reagents:
- <a name="Taq DNA polymerase 10X buffer with MgCl2">Taq DNA polymerase 10X buffer with MgCl2
<tab>(diluted 1:10 in ddH2)</a> - <a name="proteinase K">proteinase K
<tab>(20 mg/ml)</a> - tail or tissue chunk
Equipment:
- Incubator
- Centrifuge
Steps:
- Tissue lysis to release DNA
Measure out 100 µl of <a href="#Taq DNA polymerase 10X buffer with MgCl2" >Taq DNA polymerase 10X buffer with MgCl2</a> into tail or tissue chunk.
Add 2 µl of <a href="#proteinase K" >proteinase K</a>. - Incubate at 50°C for 3 - 12 hrs(overnight) with shaking 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
Incubate at 95°C for 10 - 20 mins with shaking at 300 rpm.
Centrifuge at a speed of 13000 rpm for 3 mins at room temperature and aspirate out 80 µl of top layer into sterile 1.5-ml microcentrifuge tube (2).
Discard bottom layer. - Storage before PCR
NOTE: If sample is/are used within 1 hr, set temperature to 4°C and use, else store it at -20°C.
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