Mouse tissue lysis for genotyping: Difference between revisions
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* incubate for 3h to o/n at | * 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 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) | ||
Revision as of 07:48, 2 May 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.
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
links
- Genotyping in Wikipedia
- PCR - how to do a PCR
- Designing primers - how to design primers
- PCR techniques - overview page
