Dual Colour Detection Protocol - 1597-KD
Dual colour Biotin/Cy3-FITC
Introduction
The FISH protocol is divided into two stages. Denaturation and Hybridisation are performed on day one. Washing and Detection are performed on day two. The metaphase spreads are normally prepared a day in advance. On day one, the DNA of the chromosomes and paints is denatured and the Hybridisation process (reannealing) takes place overnight. On day two the slides are washed to remove unbound DNA sequences followed by detection, counterstaining and mounting.
A PDF version of this protocol can be found here:- Dual Colour Detection Protocol
Denaturation and Hybridisation (This product is for research use only)
Approx time:
Slide Preparation: Preparation 20 min
Procedure 70 min
| Requirements (not provided) | |
|---|---|
| Equipment | Reagents |
| Coverslips | NaCl |
| Coplin jars | Na citrate |
| Humidified chamber | Double distilled water |
| Micro-pipette 1µl, 10µl, 500µl | HCl |
| Pipette 10ml, 20ml | Clear nail varnish |
| Parafilm | |
| Vortex | |
| Micro-centrifuge | |
| 45°C Water bath | |
| 37°C Incubator | |
| Fluorescence microscope with a suitable filter set | |
| Kit components: | |
|---|---|
| Detection reagent F1 | |
| Detection reagent F2 | |
| Detection reagent Y1 | |
| Blocking Protein BP | |
| Mountant (Antifade) + DAPI MD | |
| Detergent DT | |
| Solutions to be prepared: | |
|---|---|
| 20X SSC | |
| 1X SSC | |
| 4X SSC | |
| Detergent wash solution | |
| Stringency wash solution | |
| Working Reagent A | |
| Working Reagent B | |
| Working Reagent C | |
| Solution: 20X SSC | |
|---|---|
| 87.6g NaCl | |
| + | 44.1g Na citrate |
| Make up to 500ml in double distilled water | |
| Adjust pH to 7.4 using concentrated HCl (before finalizing water volume). Aliquot and autoclave | |
| Solution: 1X SSC | |
|---|---|
| 25ml 20X SSC | |
| + | 475ml Double distilled water |
| 500ml 1X SSC. Mix well | |
| Solution: 4X SSC | |
|---|---|
| 100ml 20X SSC | |
| + | 400ml Double distilled water |
| 500ml 4X SSC. Mix well | |
| Detergent wash solution: | |
|---|---|
| 500ml 4X SSC | |
| + | 250µl Detergent DT |
| 500ml Detergent wash solution. Mix well | |
| Stringency wash solution: | |
|---|---|
| 50ml Formamide | |
| + | 50ml 1XSSC |
| 100ml Stringency wash solution. Mix well | |
| Note: Stringency wash solution can be reused up to 5 times but should be discarded after 2 months | |
| Working Reagent A: | |
|---|---|
| 370µl Blocking Protein BP | |
| + | 2130µl Detergent wash solution |
| 2500µl Diluted Blocking Protein (15%) | |
| Working Reagent B: | |
|---|---|
| 6µl Detection reagent F1 | |
| + | 1244µl Working reagent A |
| 1250µl Diluted Detection reagent F1 (1:200) | |
| Note: Incubate in the dark for 5 min. Microcentrifuge at 11.000g, for 5 min | |
| Working Reagent C: | |
|---|---|
| 12.5µl Detection reagent F2 | |
| 2.5µl Detection reagent Y1 | |
| + | 1235µl Working reagent A |
| 1250µl Diluted Detection reagent F2 (1:100); Y1 (1:500) | |
| Note: Incubate in the dark for 5 min. Microcentrifuge at 11.000g, for 5 min. | |
Note: All volumes of the working reagents are for ten slides.
Procedure:
Washing
- Pre-warm solutions to 45°C in water bath at least 30 min before starting:
- Two Coplin jars of Stringency wash solution (50ml each)
- Two Coplin jars of Solution 1XSSC (50ml each)
- One Coplin jar of Detergent wash solution (50ml)
Note: The temperature is important. Check the temperature of the solutions in the Coplin jar and not of the water in the water bath. - Take out the slide from the incubator and leave in Solution 1XSSC for 5 min. Take off rubber cement and replace in Solution 1XSSC to remove the coverslip.
Note: Do not allow the slide to dry.Stringency washes:
- Wash slides twice by incubating 5 min each time in Stringency wash solution (45°C).
- Wash slides twice by incubating 5 min each time in Solution 1XSSC (45°C).
- Incubate slide 3 times for 4 min in Detergent wash solution (45°C).
Detection
- Apply 100µl of Working Reagent B onto the slide and cover with Parafilm immediately.
- Incubate slide in a humidified chamber for 15-20min at 37°C.
- Remove Parafilm from the slide and wash 3 times for 4 min each time in the Detergent wash solution at room temperature.
- Apply 100µl of Working Reagent C onto the slide and cover with Parafilm immediately.
- 8. Incubate slide in a humidified chamber for 15-20 min at 37°C
- Remove Parafilm from the slide and wash 3 times 4 minutes in Detergent wash solution at room temperature by emptying and refilling the Coplin jar
- Drain slide well and mount with 25-50µl of Reagent MD (25µl for 22x22 coverslip; 50µl for 22x50 coverslip).
- Apply glass coverslip and seal with nail varnish. Store slides in the dark at 4°C.
Note: You get almost no air bubbles when Reagent MD is applied on the coverslip and the almost dry (but not dried out!) slide is laid down on the coverslip. - View slides using standard epifluorescence filters for FITC andCy3 for counterstain DAPI.
