Difference between revisions of "Preparation of Tail Samples (for Genotyping)"
From Bridges Lab Protocols
(→PBND Solution: Tail Lysis Buffer) |
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3. Add 37% HCl drop-wise to adjust pH to 8.3. | 3. Add 37% HCl drop-wise to adjust pH to 8.3. | ||
4. Add ddH20 to 250ml. | 4. Add ddH20 to 250ml. | ||
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==Protocol== | ==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) | + | # 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 55 degrees (16 hours - O/N) | ||
# Incubate at 85 degrees for 60 min | # Incubate at 85 degrees for 60 min | ||
Revision as of 13:56, 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
- 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
