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  Section: Biotechnology Methods » Molecular Biology
 
 
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Staining Chromosomes (G-Banding)

 
     
 
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

Method

To stain metaphase chromosomes with Giemsa or Leishman’s stain to elicit a banding pattern throughout the chromosome arms, designated G-Bands. This G-Banding technique requires a chromosomal pretreatment step of trypsin to induce chromosome bands.


Time Required

30 to 60 minutes


Special Reagents
  • Leishman stain
  • Gurr buffer tablets

Procedure
  1. Prepare the staining solution the day prior to use. Also, slides should be aged at least 7–10 days or placed in a 55°C–65°C oven for 45 minutes before staining, to ensure excellent banding patterns. Aging the slides helps to eliminate fuzzy banding and increases contrast of the bands.
  2. Exact timing is important; therefore, a maximum of 5 slides should be stained at one time. Optimum time in the stain appears to be between 2.5–4 minutes. It is necessary to determine the approximate staining times for each bottle of stain solution. The exact time will vary by several seconds depending on the source of cells, age of slides, the cell concentration on the slide, etc. (refer to the table below)



    These are approximate times, and test slides need to be done to determine trypsin
    and staining time for each cell line.

  3. Mix 1 mL 0.25% stock trypsin with 49 mL 0.85% NaCl (working salt solution). Wait 4 minutes before beginning to stain to allow the trypsin to dissolve.
  4. Dip oven-dried slides that have cooled to room temperature in the trypsin solution for 5–30 seconds. The time in trypsin is dependent on slide preparation conditions, harvesting conditions, material being banded, etc. Stain a test slide first to determine optimum conditions.
  5. Rinse slides in 50-mL working salt solution.
  6. Use a graduated cylinder to mix 15 mL Leishman stain and 45 mL Gurr buffer just prior to staining, and pour into a coplin staining jar. Stain the slides for 3 to 4 minutes.
  7. Rinse slides in distilled water and air dry with compressed air or use a blow dryer.
  8. Check the slides using a Zeiss Microscope, 100X plan-apochromatic oil objective, brightfield. See “brightfield photography”.
    • Overtrypsinized chromosomes appear fuzzy; somewhat difficult to recognize exact bands.
    • Undertrypsinized chromosomes will have indistinct bands, decreased contrast; very difficult or impossible to determine bands.
    • Adequately trypsinized chromosomes will show telomeres not overly digested and G-Bands will appear sharp and in contrast.
  9. If slides are undertreated with trypsin, destain the slides before rebanding. Quickly dip the slides in 3:1 ethanol:acetic acid 2 or 3 times, or until all stain is removed. Rinse in distilled water, air dry, and reband.
  10. Coverslip with 24 × 50 mm #1 coverslip using permount.
  11. Transfer slides to a microslide box or suitable storage container.

Solutions
  • Trypsin (1:250) USP Grade; porcine parvovirus tested. Prepare a 0.25% (1X) solution by dissolving 0.25 g trypsin in 100 mL PBS. Aliquot into 1 mL quantities and store at –20°C for up to 1 year. Thaw just before using. Lowering thetemperature of the trypsin in the working salt solution will lower its activity, thus requiring longer exposure times and also increasing the life of the solution. Test slides should be run to determine optimum exposure times.
  • Gurr Buffer Solution: (1 liter). 1 tablet is dissolved in 1 liter distilled water. Produces a solution of pH approximately 6.8 at 20°C. Use solution at room temperature.
  • Working Salt Solution: (1 liter). Dissolve 8.5 g of NaCl in 1 liter distilled water. Use at room temperature.
  • Leishman Stain Solution. Dissolve 1 g Leishman stain into a 500-mL bottle of methanol. Stir for 4 hours and allow stain to age at least 1 day prior to use. If stain is not dissolved, filter stain through a 0.45-µm filter.
 
     
 
 
     




     
 
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