Reagents for in-situ hybridization

A. Prehybridization

1) Ethanol

% of EtOH	Abs.EtOH (ml)	DDW (ml)	Total volume (ml)
95	237.5	12.5	250
80	200	50	250
70	175	75	250
50	125	125	250
30	75	175	250

2) PK

PK (20 mg/ml)	20 u1
TE (pH 8.0)	20 ml

3) 10X Transcription buffer

400 mM Tris pH 8.25, 60 mM MgCl2, 20 mM spermidine (MW 145.2)

1 M Tris pH 8.25	4.0 ml
1 M MgCl2	0.6 ml
spermidine	0.029 g

Up to 10 ml, DEP autoclave

4) 0.2 M DTT/0.1 M DTT

DTT	1.545 g
DEP water	10 ml

Filter sterilize

5) 2.5 mM GTP/ATP/CTP pH 8.0

l00 mMATP	20 ul
l00 mMGTP	20 ul
l00 mMCTP	20 ul
DEP water	732 ul
1 M Tris pH 8.0	8 ul
Total volume	800 ul

Aliquot and store at -20 oC

 

6) 80 mM sodium bicarbonate, 120 mM sodium carbonate

NaHCO3	0.672 g
Na2CO3	0.1272 g
DEP water	10 ml

Aliquot and store at -20 oC

 

7) 6 M ammonium acetate pH5.2 (MW 77.08)

NH4Ac

92.5 g

Add 100 ml water to dissolve, pH to 5.2 and make to 200 ml, DEP, autoclave

 

8) Column buffer

10 mM Tris pH8, 1 mM EDTA, 10 mM DTT, 0.1% SDS

1 M Tris pH 8	2 ml
0.5 M EDTA pH 8	400 ul
1 M DTT	1 ml
DDW	194 ml

DEP, autoclave (add filtered DTT after autoclave)

 

9) Yeast tRNA (10 mg/ml)

Sigma R-8759 RNA Transfer, from Bakers Yeast, type X-SA

Dissolve power in DEP water, aliquot and store at -20 oC

 

10) 10X Saline

NaCl	33.2 g
DDW	up to 300 ml

DEP overnight and autoclave

 

11) 20X PBS

NaCl	198.9 g
KCl	5.06 g
Na2HPO4.2H2O	14.24 g
KH2PO4	6.54 g
DDW	900 ml

pH to 7.2 and top up volume to 1 litre

 

12) TE

50 mM Tris pH 8, 5 mM EDTA

1 M Tris pH 8	10 ml
500 mM EDTA	2 ml
DDW	188 ml

DEP and autoclave

 

13) 20X SSC

NaCl	175.3 g
Na citrate	88.2 g
DDW	800 ml

Adjust pH to 7.0 and top up to 1 litre

 

14) 5X NTE

0.5 M NaCl, 10 mM Tris pH 8.0, 5 mM EDTA

Tris	12.11 g
EDTA	18.612 g
NaCl	292.5 g

Adjust pH to 8.0 and top up to 2 litres

 

15) 10X Triethanolamine HCl pH 8.0

Triethanolamine

74.28 g

Adjust pH to 8.0, top up to 400 ml DDW, DEP and autoclave

 

16) 5 M NaCl

NaCl	116.8 g
DDW	400 ml

DEP and autoclave

 

17) Formamide deionized

Formamide	500 ml
Resin	20 g

Stir slowly for 25 minutes, filter with sintered glass filter, store at 4 oC in dark

 

B. Post-hybridization

1. Ethanol with ammonium acetate

% EtOH	Abs. EtOH (ml)	6 M NH4Ac (ml)	DDW (ml)	Total (ml)
30 %	120	20	260	400
60%	240	20	140	400
80 %	320	20	60	400
95 %	380	20	0	400

 

2. 50 % formamide, 5X SSC

Non-deionized Formamide	30 ml
20 X SSC	15 ml
DDW	15 ml

 

3. 5X SSC, 10 mM DDT

20X SSC	87.5 ml
2 M DTT	1.75 ml
DDW	260. 75 ml
Total	350 ml

 

4. 50 % formamide, 2X SSC, 20 mM DTT

Deionized formamide	50 ml
20 X SSC	10 ml
2 M DTT	1 ml
DDW	39 ml
Total	100 ml

 

5. RNase

RNase A (Worthington)	80 ul (final 20-25 ug/ml)
RNase T (Boehringer)	8 ul (final 50 unit/ml)
1X NTE	80 ml

 

6. 2X SSC

20X SSC	35 ml
DDW	315 ml
Total	350 ml

 

7. 0.1X SSC

20X SSC	1.75 ml
DDW	348.25 ml
Total	350 ml

 

C. Slide development

Developer: One part (100 ml) of Ilford Phensiol developer is added to 4 parts (400 ml) of distilled water

Stop solution: 2.5 ml glacial acetic acid is added to 500 ml distilled water (0.5 % acetic acid)

Fixer: 120 g sodium thiosulphate is added to 400 ml distilled water (30 % sodium thiosulphate)

Scott's tape water: 3.5 g sodium bicarbonate and 20 g magnesium sulphate is added to 1 litre of distilled water