Mouse tissue lysis for genotyping

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(proteinase K inactivation)
(material, prot K stability)
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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.
-
==== tissue lysis to release DNA ====
+
== 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)
 +
 
-
* 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
* incubate for 3h to o/n at 56°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 ====
+
=== proteinase K inactivation ===
* 95°C for 10-20 min at 300 rpm
* 95°C for 10-20 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
-
==== storage before PCR ====
+
=== storage before PCR ===
* 4°C short term, -20°C long term
* 4°C short term, -20°C long term
-
=== links ===
+
== links ==
* [http://en.wikipedia.org/wiki/Genotyping Genotyping in Wikipedia]
* [http://en.wikipedia.org/wiki/Genotyping Genotyping in Wikipedia]

Revision as of 09:48, 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.

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 56°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

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