Lab Equipment Reagents

Lab Equipment- Reagents

Materials Required

 

The following auxiliary materials, reagents and equipment are required to perform FISH assays.

  • 100% Ethanol (EtOH)
  • purified water (distilled or deionized)
  • 12N HCl (for adjusting pH of wash solutions)
  • 1N NaOH (for adjusting pH of wash solutions)
  • 20X SSC (500 g) (Vysis Order No. 32-804850)
  • NP-40 (Vysis Order #32-804818)
  • Glass Coplin jars
  • Calibrated thermometer
  • DAPI I counterstain (Vysis Order #30-804830)
  • DAPI II counterstain (Vysis Order #30-804831)
  • Forceps
  • Formamide, ultrapure grade (Vysis Order No. US75828)
  • Graduated cylinder 1000 mL
  • Magnetic stirrer
  • Methanol (for slide preparation)
  • Microcentrifuge
  • Microliter pipette tips for 1-10 µL volumes
  • Microliter pipettor for 1-10 µL capacity
  • pH meter
  • Pre-cleaned glass microscope slides
  • Purified water
  • Timer
  • Vortex mixer
  • Water bath (37 and 72 °C)

Pretreatment Reagents

  • Paraffin Pretreatment Kit I (32-801200)
  • Paraffin Pretreatment Kit II (32-801210)
  • Paraffin Pretreatment Kit III (32-801230)

  

 
 
 
 
Preparing the Reagents

Note: Where indicated, measure the pH of these solutions at ambient temperature. Use a pH meter with a glass electrode unless otherwise noted.

 

20X SSC solution
Mix thoroughly 132g 20X SSC in 400 mL purified H2O. Measure pH and adjust to pH 5.3 with HCl. Add purified H2O to bring final volume to 500 mL. Store at ambient temperature. Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated.

 

 2X SSC solution
Mix thoroughly 100 mL 20X SSC (pH 5.3) with 850 mL purified H2O. Measure pH and adjust to pH 7.0 ±0.2 with NaOH. Add purified H2Oto bring final volume to 1 liter. Store at ambient temperature. Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated.

 

Denaturation Solution (70% Formamide/2X SSC)
Mix thoroughly 49 mL ultrapure formamide, 7 mL 20X SSC (pH 5.3) and 14 mL purified H2O in a glass Coplin jar. Measure pH using pH indicator strips to verify pH is 7.0-8.0. Between uses, store covered at 2-8 °C. Discard after 7days.

 

0.4X SSC/0.3% NP-40 Wash Solution
Mix thoroughly 20 mL 20X SSC (pH 5.3) with 950 mL purified H2O.Add 3 mL of NP-40. Mix thoroughly until NP-40 is completely dissolved. Measure pH and adjust pH to 7.0-7.5 with NaOH. Add purified H2O to bring final volume of the solution to 1 liter. Store at ambient temperature. Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated.

 

2X SSC/0.1% NP-40 Wash Solution
Mix thoroughly 100 mL 20X SSC (pH 5.3) with 850 mL purified H2O. Add 1 mL NP-40. Measure pH and adjust to pH 7.0 ±0.2 with NaOH. Add purified H2Oto bring final volume to 1 liter. Store at ambient temperature. Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated.

 

Ethanol Solutions (70%, 85%, 100%)
Prepare v/v dilutions of 100% ethanol with purified H2O. Between uses, store tightly covered at ambient temperature. Discard stock solutions after 6 months.

 

PBS
Prepare PBS by diluting 20 mL of PBS 10X with 180 mL of purified H2O. Store at ambient temperature. Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated.

 

RNase A Stock and Working Solutions
Prepare a stock solution of 10 mg/ mL RNase A (DNase free) in 2X SSC. If RNase is not labeled as DNase free, then place RNase solution in boiling water bath for 10 minutes to inactive DNase. Aliquot into 10 µL volumes and store at -20 °C in capped 1.0 mL tubes. At time of use, prepare working RNase solution by thawing the stock RNase solution (10mg/mL) in the tube and directly adding 990 µL of 2X SSC pH 7.0-7.4 tothe 10 µL contained within the tube. Vortex to mix. Do not freeze-thaw. Discard after use.

 

Pepsin Stock and Working Solutions
Prepare a 10% Pepsin stock solution by mixing 0.1 gram pepsin in 1 mL water (pre-warmed to 37 °C). Aliquot into 35 µL volumes and store at -20 °C until ready to use. When ready to use prepare a 0.005% pepsin working solution by mixing 35 µL of the 10% pepsin stock into solution of 69.3mL purified water and 0.7 mL 1.0 N HCl that has been heated to 37 º C in Coplin jar ( using a 37 °C water bath).

 

Formaldehyde Fixation Solution
Mix together 57.6 mL of purified water, 3.5 mL of 1M MgCl2, 7 mL of 10X PBS and 1.89 mL of 37% formaldehyde. Mix well. Place in a Coplin jar. Prepare fresh each day.

 
 
 
 

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