Extraction of polytene chrmosome
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PREPARATION OF SALIVARY GLAND CHROMOSOMES OF LARVA OF DROSOPHILA
AIM: To prepare temporary mount of salivary gland chromosomes of larva of Drosophila
MATERIALS REQUIRED: Rava, Jaggery, Agar-Agar, Bottles, Cotton plug, Glass rod, Propicnic acid, third instar larvae, Needle, Forceps, Slide, Ringers fluid , lacto acetocaramine ,HCl, dissecting microscope, blotting paper, coverslip and compound microscope
PROCEDURE:
1. PREPARATION OF MEDIUM
Ø Bottles should be thoroughly washed and rinse with detergents.
Ø Bottles have to be sterilized by autoclaving it at 80 degree Celsius for twenty–four hours.
Ø Take one litre of water in vessel and add 100g of Rava, 2g of agar-agar and Jaggery into it.
Ø Boil it for few minutes.
Ø Add 2 ml of propicnic acid to the mixture to avoid fungus attack.
Ø Boil till it becomes thick. (For 15 minutes).
Ø Transfer it to the bottles later.
Ø Close the mouth with the cotton and leave it for four for solidifying.
Ø After one day wipe the water droplets present inside the bottle with the help of blotting sheet.
Ø Transfer the fruit flies in the bottles.
Ø Label date, time and your name on bottle and add two drops of yeast solution into the bottle.
Ø Keep it in incubator at temperature 21 degree Celsius for three days.
2. MOUNTING POLYTENE CHROMOSOME
· Take a clean slide and keep it on dissecting microscope.
· Put a drop of ringer’s fluid onto the slide.
· Take out biggest size third instar larvae onto the slide.
· The third instar larva is held by a forcep and with the help of needle.
· The jaws are squeeze out from the alimentary canal by holding middle body of the larvae with one needle; another needle is put on jaws and slowly pulls out the jaws in the opposite direction.
· Separate the salivary gland from digestive system.
· Take a clean slide, put a drop of lacto acetocaramine stain on it.
· Carefully transfer the salivary glands on the slide with the help of needle or pointed brush.
· Keep it undisturbed for 20 minutes.
· After 20 minutes, using blotting paper, remove the stain, add a drop of 1 N HCl and put a coverslip on it.
· Cover the slide with blotting paper and gently tap on coverslip using blunt end of the pencil to make squash of salivary gland.
· Observe under the compound microscope, if polytene chromosomes are seen, immediately seal the slide with wax.