Difference between revisions of "Preparation of Tail Samples (for Genotyping)"

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(PBND Solution: Tail Lysis Buffer)
(changed g to ml for detergents)
 
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* 0.025g 0.1 mg/mL MgCl2, 6H20
 
* 0.025g 0.1 mg/mL MgCl2, 6H20
 
* 0.025g 0.1 mg/mL Gelatin
 
* 0.025g 0.1 mg/mL Gelatin
* 1.125g 0.45% (NP-40) Jgepal  
+
* 1.125ml 0.45% (NP-40) Jgepal  
* 1.125g 0.45% Tween 20
+
* 1.125ml 0.45% Tween 20
  
  

Latest revision as of 15:20, 21 May 2018

PBND Solution: Tail Lysis Buffer

  • 50mM KCl (Hint: KCl is very heavy, you'll only need a tiny bit)
  • 10mM Tris HCl (pH~8.3)
  • 0.1 mg/mL MgCl2, 6H20
  • 0.1 mg/mL Gelatin
  • 0.45% (NP-40) Jgepal (Hint: Use cut tips, as NP-40 is very viscous)
  • 0.45% Tween 20 (Hint: Use cut tips, as Tween 20 is very viscous)

PBND can be made in liter volume, aliquoted into 50ml falcon tubes, frozen, and used as needed

For 250ml PBND:

  • 0.930g 50mM KCl
  • 0.300g 10mM Tris HCl (pH~8.3)
  • 0.025g 0.1 mg/mL MgCl2, 6H20
  • 0.025g 0.1 mg/mL Gelatin
  • 1.125ml 0.45% (NP-40) Jgepal
  • 1.125ml 0.45% Tween 20


  1. Start by adding 150ml ddH2O to a beaker on a stir plate (with magnetic stir bar).
  2. Add reagents. Mix well.
  3. Add 37% HCl drop-wise to adjust pH to 8.3.
  4. Add ddH20 to 250ml.

Protocol

  1. Combine 100uL of PBND solution with 2ul of Proteinase K and mouse tail in an eppendorf tube or in a well of a 96 well PCR plate (Proteinase K stock = 10mg/mL in ddH2O)
  2. Incubate at 55 degrees (16 hours - O/N)
  3. Incubate at 85 degrees for 60 min
  4. Hold at 4 degrees