==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)
ProtocolPBND 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 0.1mg/mL MgCl2, 6H20* 0.025g 0.1 mg/mL Gelatin* 1.125ml 0.45% (NP-40) Jgepal * 1.125ml 0.45% Tween 20 # Start by adding 150ml ddH2O to a beaker on a stir plate (with magnetic stir bar).# Add reagents. Mix well.# Add 37% HCl drop-wise to adjust pH to 8.3.# Add ddH20 to 250ml. ==Protocol==# Combine 200uL 100uL of PBND solution with 2ul of Proteinase K and mouse tail in an eppindorf eppendorf tube2.or in a well of a 96 well PCR plate (Proteinase K stock = 10mg/mL in ddH2O) # Incubate at 55 degrees (3hrs16 hours -O/N)3.) # Incubate at 85 degrees for 45 60 min # Hold at 4.) PCRdegrees [[Category: Mouse Work]][[Category: Molecular Biology]][[Category: DNA]][[Category: Genotyping]]