Difference between revisions of "Preparation of Tail Samples (for Genotyping)"
From Bridges Lab Protocols
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− | PBND Solution: Tail Lysis Buffer | + | ==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 | + | * 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 | |
− | 1.) Combine | + | |
− | + | For 250ml PBND: | |
− | + | * 0.930g 50mM KCl | |
− | 4 | + | * 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 | ||
+ | |||
+ | |||
+ | # 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 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) | ||
+ | # Incubate at 55 degrees (16 hours - O/N) | ||
+ | # Incubate at 85 degrees for 60 min | ||
+ | # Hold at 4 degrees | ||
+ | |||
+ | |||
+ | [[Category: Mouse Work]] | ||
+ | [[Category: Molecular Biology]] | ||
+ | [[Category: DNA]] | ||
+ | [[Category: Genotyping]] |
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
- 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 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)
- Incubate at 55 degrees (16 hours - O/N)
- Incubate at 85 degrees for 60 min
- Hold at 4 degrees