Chromosome preparation

• Home
• People
• Publications
• Research Projects
• Protocols
• Useful Journals
• Useful Links

              Chromosome Preparation for In Situ Hybridization

Squash Method

1.     Germinate seeds on moist filter paper in petri dishes at room temperature.

2.     Root-tips at 1-2 cm long are collected and placed in vials with water.  Place the vials in a box with crushed ice in water and store box in a refrigerator for 22-24 hrs.

3.     Root tips fixed in a 3:1 mixture of 95% ethanol:glacial acetic acid for 1-7 days.

        Notes: better in situ hybridization results will be obtained if the fixation is not too long.  However, it is always relatively difficult to make preparation if the fixation is less than two days.  Try your best.

4.     Stain the root tips in 1% acetocarmine for 10 min - 1 hr.  Do not stain the root tips too long.

5.     Make the preparation the way you like.  But never try to soften the root tips by warming, or even boiling them.

6.     Store the slide in a -70°C freezer.  The slides can be stored up to years without seriously affecting the N-banding and in situ hybridization results in our hands.

Notes: Many experienced cytogeneticists love to say that making preparations is the most important step for any cytogenetic or molecular cytogenetic techniques.  This is true for in situ hybridization.  We introduce you to a very simple way to check the quality of your preparations for in situ hybridization.  If the morphology of the chromosomes in most of the cells does not change (under a phase contrast microscope) before and after the ethanol series treatment (for drying), it means you are doing a good job.

Maceraction Method