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  Section: Biotechnology Methods » Molecular Biology
 
 
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Salivary Gland Preparation (Squash Technique)

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method

Materials
  • Fruit fly larva (wild-type and tandem-duplication mutants)
  • Ringer’s insect saline
  • Fine forceps and probe
  • Aceto-orcein
  • Dissecting and regular microscopes
  • Slides, coverslips
  • Small dish of melted paraffin
  • Paintbrush
Procedure
  1. Select a third instar larva, for which the cuticle has not yet hardened, from a wild-type culture of Drosophila. Place it into a drop of Ringer’s saline solution on a slide.
  2. Place the slide on the stage of a dissecting microscope and view the larva with low power. Grasp the anterior of the larva with a fine-point forceps and pin down the posterior portion with a probe. Gently pull the head off and discard the tail of the larva.
  3. Locate the salivary glands and their attached fat bodies. The glands are semitransparent and attached by ducts to the digestive system. The fat bodies are white and opaque. Tease away the fat bodies and discard.
  4. Place a drop of aceto-orcein on the slide next to the Ringer’s and move the salivary glands into the stain. Blot away any excess Ringer’s.
  5. Place a coverslip over the preparation and allow it to stand for 1–3 minutes (it will take a few trials to obtain properly stained chromosomes). Gently squash the gland preparation in the following manner:
    • Place the slide between several layers of paper toweling.
    • Place your thumb on the top of the towel immediately over the coverslip and gently roll your thumb while exerting a small amount of pressure (as though you were making a fingerprint). Do not move your thumb back and forth. One gentle roll is sufficient.
    • Remove the slide from the towels, and seal the edges of the coverslip by using a paintbrush dipped in melted paraffin.
  6. Examine the slide with the microscope and diagram the banding patterns that are observed.
  7. Repeat the squash technique using larva from a genetic variant known to be the result of a deletion and/or tandem duplication. Determine the location of the deleted or duplicated bands on the chromosomes.
 
     
 
 
     




     
 
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