Feb 01, 2023
Preparation of Buffers for PhageFISH protocol
Peer-reviewed method
- Line Jensen Ostenfeld1,
- Saria Otani1
- 1DTU
Protocol Citation: Line Jensen Ostenfeld, Saria Otani 2023. Preparation of Buffers for PhageFISH protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpjop8gzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 27, 2023
Last Modified: February 01, 2023
Protocol Integer ID: 75961
Keywords: Permeabilisation buffer, rRNA hybridisation buffer, rRNA hybridisation wash buffer, rRNA CARD buffer, Gene hybridisation buffer, Gene hybridisation wash buffer
Funders Acknowledgement:
NovoNordisk
Grant ID: NNF16OC0021856
Abstract
This protocol details about preparation of various buffers for PhageFISH protocol.
Attachments
627-1301.docx
32KB
Materials
Materials and Reagents
- PBS [pH 7.5] (10 x)
- Tris-HCl [pH 8.0](1 M)
- EDTA (0.5)
- Water
- Lysozyme
- Dextran sulphate
- NaCl (5 M)
- Tris-HCl [pH 8.0] (1 M)
- Nucleic acid blocking reagent (10%)
- Sheared salmon sperm (10 mg/ml)
- Yeast RNA (10 mg/ml)
- Formamide (100%)
- SDS (20%)
- SSC (20 x)
- 0.22µm syringe filter
- 50 ml falcon tube
Permeabilisation buffer
Permeabilisation buffer
50 ml:
| A | B | |
| PBS [pH 7.5] (10 x) | 5 ml | |
| Tris-HCl [pH 8.0] (1 M) | 5 ml | |
| EDTA (0.5) | 5 ml | |
| Water | 35 ml | |
| Lysozyme |
Permeabilisation mix: Mix PBS, Tris-HCl, EDTA, and water.
High conc. lysozyme buffer: Dissolve lysozyme in appropriate buffer volume (50 mg lysozyme to 10 mL buffer). It may be necessary to heat the solution to 37 °C .
Dilute lysozyme buffer into large buffer volume by adding 1 part lysozyme buffer to 9 parts permeabilisation mix.
Final concentration:
| A | B | |
| PBS | 1 x | |
| Tris-HCl | 0.1 M | |
| EDTA | 0.05 M | |
| Lysozyme | 0.5 mg/ml |
Note
- Permeabilisation buffer should not be stored.
- Prepare in aliquots of 1 mL .
rRNA hybridisation buffer
rRNA hybridisation buffer
40 ml:
| A | B | |
| Dextran sulphate | 4 g | |
| NaCl (5 M) | 7.2 ml | |
| Tris-HCl [pH 8.0] (1 M) | 0.8 ml | |
| Water | 4 ml | |
| Nucleic acid blocking reagent (10%) | 4 ml | |
| Sheared salmon sperm (10 mg/ml) | 1 ml | |
| Yeast RNA (10 mg/ml) | 1 ml | |
| Formamide (100%) | 17.5 ml | |
| SDS (20%) | 40 µl |
Mix dextran sulphate, NaCl, Tris-HCl, and water in a falcon tube and vortex or shake thoroughly to disperse dextran sulphate. Heat solution in waterbath at 37-48 °C and vortex until dextran sulphate is completely dissolved.
Cool the solution down to Room temperature .
Add nucleic acid blocking agent, sheared salmon sperm, yeast RNA, formamide, and SDS. Adjust volume with water to reach 40 mL if necessary.
Vortex to mix.
Spin down solution briefly and filter through 0.22µm syringe filter.
Final concentration:
| A | B | |
| Dextran sulphate | 10% | |
| NaCl | 0.9 M | |
| Tris-HCl | 20 mM | |
| Nucleic acid blocking reagent | 1% | |
| Sheared salmon sperm | 0.25 mg/ml | |
| Yeast RNA | 0.25 mg/ml | |
| Formamide | 35% | |
| SDS | 0.02% |
Store in aliquots at -20 °C . Reheat to 37 °C before use to redissolve precipitate.
Prepare several in aliquots of 900 µL .
rRNA hybridisation wash buffer
rRNA hybridisation wash buffer
50 ml:
| A | B | |
| *NaCl (5 M) | 700 µl | |
| *EDTA [pH 8.0] (0.5 M) | 500 µl | |
| Tris-HCl (1 M) | 1 ml | |
| Water | up to 50 ml | |
| SDS (20%) | 25 µl |
Mix *NaCl, *EDTA, and Tris-HCl in 50 ml falcon tube.
Note
* NOTE: Na+ concentrations depend on the amount of formamide used in the hybridisation buffer. The formamide concentration is calculated based on probe properties to achieve a hybridisation temperature of 42-50 °C .
Add water up the 50 ml mark.
Add SDS.
Final concentrations:
| A | B | |
| NaCl | 70 mM | |
| EDTA | 5 mM | |
| Tris-HCl | 20 mM | |
| SDS | 0.01% |
The formamide (FA) concentrations and the corresponding Na+ ions concentrations when washing at 48 °C are as follows:
| A | B | |
| 0% FA | 900 mM Na+ | |
| 5%FA | 636 mM Na+ | |
| 10% FA | 450 mM Na+ | |
| 15% FA | 318 mM Na+ | |
| 20% FA | 225 mM Na+ | |
| 25% FA | 159 mM Na+ | |
| 30% FA | 112 mM Na+ | |
| 35% FA | 80 mM Na+ | |
| 40% FA | 56 mM Na+ | |
| 45% FA | 40 mM Na+ | |
| 50% FA | 28 mM Na+ | |
| 55% FA | 20 mM Na+ | |
| 60% FA | 14 mM Na+ |
Prepare in aliquots of 50.
Note
Prepare at least two aliquots per cycle.
rRNA CARD buffer
rRNA CARD buffer
40 ml:
| A | B | |
| Dextran sulphate | 4 g | |
| PBS [pH 7.4] (10 x) | 4 ml | |
| NaCl (5 M) | 16 ml | |
| Water | up to 40 ml | |
| Nucleic acid blocking reagent (10%) | 400 µl |
Mix dextran sulphate, PBS, and NaCl. Add water up to 40 ml. vortex thoroughly to disperse dextran sulphate. Heat solution in waterbath at 37-48 °C and vortex until dextran sulphate is completely dissolved.
Allow solution to cool down to room temperature and add nucleic acid blocking reagent.
Vortex to mix.
Spin down briefly.
Filter through 0.22 µm syringe filter.
Final concentration:
| A | B | |
| PBS | 1x | |
| Dextran sulphate | 10% | |
| Nucleic acid blocking reagent | 0.10% | |
| NaCl | 2 M |
Store in aliquots at 4 °C . Reheat to 37 °C before use to redissolve precipitate.
Prepare in aliquot of 3 mL .
Gene hybridisation buffer
Gene hybridisation buffer
40 ml:
| A | B | |
| Dextran sulphate | 4 g | |
| SSC (20 x) | 10 ml | |
| EDTA [pH 8.0] (0.5 M) | 1.6 ml | |
| Water | 4.4 ml | |
| Nucleic acid blocking reagent (10%) | 4 ml | |
| Sheared salmon sperm (10 mg/ml) | 1 ml | |
| Yeast RNA (10 mg/ml) | 1 ml | |
| Formamide (100%) | 14 ml | |
| SDS (20%) | 200 µl |
Mix dextran sulphate, SSC, EDTA, and water in a falcon tube and vortex or shake thoroughly to disperse dextran sulphate. Heat solution in waterbath at 37-48 °C and vortex until dextran sulphate is completely dissolved.
Cool the solution down to Room temperature .
Add nucleic acid blocking agent, sheared salmon sperm, yeast RNA, formamide, and SDS.
Vortex to mix.
Spin down solution briefly and filter through 0.22 µm syringe filter.
Final concentration:
| A | B | |
| Formamide | 35% | |
| SSC | 5x | |
| Dextran sulphate | 10% | |
| SDS | 0.10% | |
| EDTA | 20 mM | |
| Nucleic acid blocking reagent | 1% | |
| Sheared salmon sperm | 0.25 mg/ml | |
| Yeast RNA | 0.25 mg/ml |
Store in aliquots at -20 °C . Reheat to 42 °C before use to redissolve precipitate.
Gene hybridisation wash buffer I
Gene hybridisation wash buffer I
50 ml:
| A | B | |
| SSC (20 x) | 5 ml | |
| SDS | 250 µl | |
| Water | up to 50 ml |
Mix SSC and water in a 50 ml falcon tube.
Add SDS.
Vortex to mix.
Final concentration:
| A | B | |
| SSC | 2 x | |
| SDS | 0.1% |
Store for 1-2 days at 42 °C .
Gene hybridisation wash buffer II
Gene hybridisation wash buffer II
50 ml:
| A | B | |
| SSC (20 x) | 250 µl | |
| SDS | 250 µl | |
| Water | up to 50 ml |
Mix SSC and water in a 50 ml falcon tube.
Add SDS.
Vortex to mix.
Final concentration:
| SSC | 0.1 x | |
| SDS | 0.10% |
Store for 1-2 days at 42 °C .
Gene CARD amplification buffer
Gene CARD amplification buffer
40 ml:
| A | B | |
| Dextran sulphate | 8 g | |
| PBS [pH 7.4] (10 x) | 4 ml | |
| NaCl (5 M) | 16 ml | |
| Water | 15.6 ml | |
| Nucleic acid blocking reagent (10%) | 400 µl |
Mix dextran sulphate, PBS, NaCl, and water.
Vortex or shake thoroughly to disperse dextran sulphate. Heat solution in waterbath at 37-48 °C and vortex until dextran sulphate is completely dissolved.
Allow solution to cool down to room temperature and add nucleic acid blocking reagent.
Vortex to mix.
Spin down briefly.
Filter through 0.22 µm syringe filter.
Final concentrations:
| A | B | |
| PBS | 1x | |
| Dextran sulphate | 20% | |
| Blocking reagent | 0.10% | |
| NaCl | 2 M |
Store in aliquots at 4 °C . Reheat to 37 °C before use to redissolve precipitate.