Difference between revisions of "Preparation of Tail Samples (for Genotyping)"
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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) |
| + | |||
| + | 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== | ==Protocol== | ||
| − | # Combine | + | # 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 (O/N) | + | # Incubate at 55 degrees (16 hours - O/N) |
| − | # Incubate at 85 degrees for | + | # 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
