Preparation of Buffers for PhageFISH protocol

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 (50mg lysozyme to 10mL 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

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 40mL 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 900µL.

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.

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.

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 3mL.

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.

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 .

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:

A	B
SSC	0.1 x
SDS	0.10%

Store for 1-2 days at 42°C.

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.