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

Revision as of 14:01, 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)

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
• 1.125g 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 in ddH2O)
2. Incubate at 55 degrees (16 hours - O/N)
3. Incubate at 85 degrees for 60 min
4. Hold at 4 degrees