Protocol for Chromosome Squashing

1. Obtain root tip sample

2. Grab tweezers and grab the root tip and place it on a microscope slide (make sure not to put the tip in the middle of the slide)

3. Immediately afterwards place one drop of 45% acetic acid on the root tip

4. Take a razor blade and as thinly as you can, cut the dark tip of the root into three very small slices

5. Put one drop of 45% acetic acid on the three small cuts

6. Using the razorblade slide the three cuts to the middle of the slide

7. Set the razor blade on the slide and put the cover slip on top of it so that the slip it not flat on the slide

8. While holding the cover slip tightly take a blunt ended object and tap the top of the cover slip several times until the root cuts almost dissipate

9. Take the razor blade out from under the slip and use a paper towel to dab the sides of the slide to soak the excess acetic acid

10. Flame the bottom of the slide twice under a Bunsen burner and immediately following flip the slide over and put as much force as you can down on the slide until you are satisfied (squashing).

11. Examine under microscope

12. When finished place slides at -20 C for storage


Protocol for Fluorescence Examination

1. Take a slide out of the -20 C freezer and place in liquid Nitrogen until cooled

2. Take slide out of Nitrogen and remove the cover slip with a razor blade

3. Place the slide in 70% Ethanol for five minutes then followed by 90% Ethanol for 5 minutes then 100% ethanol for minutes

4. Drain the excess ethanol from the slide by putting the edge on a paper towel and letting the liquid drain

5. To analyze the slide, put 20 micro liters of Propidium Iodide (PI) on the sample and cover it with a 24x33mm cover slide

6. Analyze under fluorescence microscope