TITLE: Preparation of Buffers for PhageFISH protocol
AUTHORS: Line Jensen Ostenfeld, Saria Otani

[DESCRIPTION]
This protocol details about preparation of various buffers for PhageFISH protocol.

[STEPS]
SECTION: Permeabilisation buffer
1. 50 ml: 
      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.
SECTION: Permeabilisation buffer
2. 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. 

SECTION: Permeabilisation buffer
3. Dilute lysozyme buffer into large buffer volume by adding 1 part lysozyme buffer to 9 parts permeabilisation mix. 
SECTION: Permeabilisation buffer
4. Final concentration:
 

 

SECTION: rRNA hybridisation buffer
5. 40 ml: 
      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.

SECTION: rRNA hybridisation buffer
6. Cool the solution down to Room temperature. 

SECTION: rRNA hybridisation buffer
7. Add nucleic acid blocking agent, sheared salmon sperm, yeast RNA, formamide, and SDS. Adjust volume with water to reach 40 mL if necessary. 

SECTION: rRNA hybridisation buffer
8. Vortex to mix. 
SECTION: rRNA hybridisation buffer
9. Spin down solution briefly and filter through 0.22µm syringe filter. 
SECTION: rRNA hybridisation buffer
10. Final concentration:
      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%  
 
SECTION: rRNA hybridisation buffer
11. Store in aliquots at -20 °C. Reheat to 37 °C before use to redissolve precipitate. 


SECTION: rRNA hybridisation wash buffer
13. 50 ml:  
      *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. 
 

SECTION: rRNA hybridisation wash buffer
14. Add water up the 50 ml mark. 
SECTION: rRNA hybridisation wash buffer
15. Add SDS. 
SECTION: rRNA hybridisation wash buffer
16. Final concentrations:  
      NaCl  70 mM    EDTA  5 mM    Tris-HCl  20 mM    SDS  0.01%  
 
SECTION: rRNA hybridisation wash buffer
17. The formamide (FA) concentrations and the corresponding Na+ ions concentrations when washing at 48 °C are as follows:  
      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+  

SECTION: rRNA hybridisation wash buffer
18. Prepare in aliquots of 50.
 

SECTION: rRNA CARD buffer
19. 40 ml: 
 
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.  

SECTION: rRNA CARD buffer
20. Allow solution to cool down to room temperature and add nucleic acid blocking reagent. 
SECTION: rRNA CARD buffer
21. Vortex to mix. 
SECTION: rRNA CARD buffer
22. Spin down briefly. 
SECTION: rRNA CARD buffer
23. Filter through 0.22 µm syringe filter. 
SECTION: rRNA CARD buffer
24. Final concentration:   
      PBS  1x    dextran sulphate  10%    nucleic acid blocking reagent  0.10%    NaCl  2 M  

SECTION: rRNA CARD buffer
26. Prepare in aliquot of 3 mL.

SECTION: Gene hybridisation buffer
27. 40 ml: 
      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. 
SECTION: rRNA hybridisation buffer
12. Prepare several in aliquots of 900 µL.
SECTION: Gene hybridisation buffer
28. Cool the solution down to Room temperature. 

SECTION: Gene hybridisation buffer
29. Add nucleic acid blocking agent, sheared salmon sperm, yeast RNA, formamide, and SDS. 
SECTION: Gene hybridisation buffer
30. Vortex to mix.
SECTION: Gene hybridisation buffer
31. Spin down solution briefly and filter through 0.22 µm syringe filter. 
SECTION: Gene hybridisation buffer
32. Final concentration:  
      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  

SECTION: Gene hybridisation wash buffer I
34. 50 ml: 
 
Mix SSC and water in a 50 ml falcon tube.
SECTION: Gene hybridisation wash buffer I
35. Add SDS. 
SECTION: Gene hybridisation wash buffer I
36. Vortex to mix. 
SECTION: Gene hybridisation wash buffer I
37. Final concentration: 
 

SECTION: Gene hybridisation wash buffer II
39. 50 ml:  
      SSC (20 x)  250 µl    SDS  250 µl    Water  up to 50 ml  
 Mix SSC and water in a 50 ml falcon tube. 
SECTION: Gene hybridisation wash buffer II
40. Add SDS. 
SECTION: Gene hybridisation wash buffer II
41. Vortex to mix. 
SECTION: Gene hybridisation wash buffer II
42. Final concentration:  
      SSC  0.1 x     SDS  0.10%  

SECTION: Gene CARD amplification buffer
44. 40 ml:  
      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. 
SECTION: Gene CARD amplification buffer
45. Vortex or shake thoroughly to disperse dextran sulphate. Heat solution in waterbath at 37-48 °C and vortex until dextran sulphate is completely dissolved. 
SECTION: Gene CARD amplification buffer
46. Allow solution to cool down to room temperature and add nucleic acid blocking reagent. 
SECTION: Gene CARD amplification buffer
47. Vortex to mix.
SECTION: Gene CARD amplification buffer
48. Spin down briefly. 
SECTION: Gene CARD amplification buffer
49. Filter through 0.22 µm syringe filter. 
SECTION: Gene CARD amplification buffer
50. Final concentrations:  
      PBS  1x    dextran sulphate  20%    blocking reagent  0.10%    NaCl  2 M  
 
SECTION: rRNA CARD buffer
25. Store in aliquots at 4 °C. Reheat to 37 °C before use to redissolve precipitate. 
SECTION: Gene hybridisation buffer
33. Store in aliquots at -20 °C. Reheat to 42 °C before use to redissolve precipitate. 

SECTION: Gene hybridisation wash buffer I
38. Store for 1-2 days at  42 °C .

SECTION: Gene hybridisation wash buffer II
43. Store for 1-2 days at 42 °C.

SECTION: Gene CARD amplification buffer
51. Store in aliquots at 4 °C. Reheat to 37 °C before use to redissolve precipitate.