Feb 01, 2023

Preparation of Buffers for PhageFISH protocol

PLOS One
Peer-reviewed method
DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpjop8gzp/v1
  • 1DTU
Saria Otani
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpjop8gzp/v1
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 , phagefish protocol, preparation of buffer, preparation of various buffer, phagefish, various buffer, buffer, protocol this protocol detail, protocol detail, protocol, preparation, phagefish protocol this protocol detail
Funders Acknowledgements:
NovoNordisk
Grant ID: NNF16OC0021856
Abstract
This protocol details about preparation of various buffers for PhageFISH protocol.
Attachments
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627-1301.docx
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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
50 ml:
AB
PBS [pH 7.5] (10 x)5 ml
Tris-HCl [pH 8.0] (1 M)5 ml
EDTA (0.5)5 ml 
Water35 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:
AB
PBS1 x
Tris-HCl0.1 M
EDTA0.05 M
Lysozyme 0.5 mg/ml

Note
  • Permeabilisation buffer should not be stored.
  • Prepare in aliquots of 1 mL .

rRNA hybridisation buffer
40 ml:
AB
Dextran sulphate4 g
NaCl (5 M)7.2 ml 
Tris-HCl [pH 8.0] (1 M)0.8 ml 
Water4 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:
AB
Dextran sulphate10%
NaCl0.9 M
Tris-HCl20 mM
Nucleic acid blocking reagent1%
Sheared salmon sperm0.25 mg/ml
Yeast RNA0.25 mg/ml
Formamide35%
SDS0.02%
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
50 ml:
AB
*NaCl (5 M)700 µl
*EDTA [pH 8.0] (0.5 M)500 µl
Tris-HCl (1 M)1 ml 
Waterup 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:
AB
NaCl70 mM
EDTA5 mM
Tris-HCl20 mM
SDS0.01%
The formamide (FA) concentrations and the corresponding Na+ ions concentrations when washing at 48 °C are as follows:
AB
0% FA900 mM Na+
5%FA636 mM Na+ 
10% FA450 mM Na+ 
15% FA318 mM Na+ 
20% FA225 mM Na+ 
25% FA159 mM Na+ 
30% FA112 mM Na+ 
35% FA80 mM Na+
40% FA56 mM Na+ 
45% FA40 mM Na+ 
50% FA28 mM Na+ 
55% FA20 mM Na+ 
60% FA14 mM Na+

Prepare in aliquots of 50.
Note
Prepare at least two aliquots per cycle.

rRNA CARD buffer
40 ml:
AB
Dextran sulphate 4 g
PBS [pH 7.4] (10 x)4 ml
NaCl (5 M)16 ml
Waterup 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:
AB
PBS1x
Dextran sulphate10%
Nucleic acid blocking reagent0.10%
NaCl2 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
40 ml:
AB
Dextran sulphate4 g
SSC (20 x)10 ml 
EDTA [pH 8.0] (0.5 M)1.6 ml  
Water4.4 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:
AB
Formamide35%
SSC5x
Dextran sulphate10%
SDS0.10%
EDTA20 mM
Nucleic acid blocking reagent1%
Sheared salmon sperm0.25 mg/ml
Yeast RNA0.25 mg/ml

Store in aliquots at -20 °C . Reheat to 42 °C before use to redissolve precipitate.

Gene hybridisation wash buffer I
50 ml:
AB
SSC (20 x)5 ml
SDS250 µl
Waterup to 50 ml
Mix SSC and water in a 50 ml falcon tube.
Add SDS.
Vortex to mix.
Final concentration:
AB
SSC2 x
SDS0.1%

Store for 1-2 days at 42 °C .

Gene hybridisation wash buffer II
50 ml:
AB
SSC (20 x)250 µl
SDS250 µl
Waterup to 50 ml
Mix SSC and water in a 50 ml falcon tube.
Add SDS.
Vortex to mix.
Final concentration:
SSC0.1 x 
SDS0.10%

Store for 1-2 days at 42 °C .

Gene CARD amplification buffer
40 ml:
AB
Dextran sulphate8 g
PBS [pH 7.4] (10 x)4 ml
NaCl (5 M)16 ml
Water15.6 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:
AB
PBS1x
Dextran sulphate20%
Blocking reagent0.10%
NaCl2 M
Store in aliquots at 4 °C . Reheat to 37 °C before use to redissolve precipitate.