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

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(Protocol)
(PBND Solution: Tail Lysis Buffer)
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==PBND Solution: Tail Lysis Buffer==
 
==PBND Solution: Tail Lysis Buffer==
* 50mM KCl
+
* 50mM KCl                           (Hint: KCl is very heavy, you'll only need a tiny bit)
 
* 10mM Tris HCl (pH~8.3)
 
* 10mM Tris HCl (pH~8.3)
 
* 0.1 mg/mL MgCl2, 6H20
 
* 0.1 mg/mL MgCl2, 6H20
 
* 0.1 mg/mL Gelatin
 
* 0.1 mg/mL Gelatin
* 0.45% (NP-40) Jgepal  
+
* 0.45% (NP-40) Jgepal       (Hint: Use cut tips, as NP-40 is very viscous)
* 0.45% Tween 20  
+
* 0.45% Tween 20                 (Hint: Use cut tips, as Tween 20 is very viscous)
 +
 
 +
PBDN 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
 +
* 1.125g  0.45% (NP-40) Jgepal
 +
* 1.125g  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==
 
==Protocol==

Revision as of 13:54, 17 August 2017

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)

PBDN 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
  • 1.125g 0.45% (NP-40) Jgepal
  • 1.125g 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)
  2. Incubate at 55 degrees (16 hours - O/N)
  3. Incubate at 85 degrees for 60 min
  4. Hold at 4 degrees