Algae, Tree, Herbs, Bush, Shrub, Grasses, Vines, Fern, Moss, Spermatophyta, Bryophyta, Fern Ally, Flower, Photosynthesis, Eukaryote, Prokaryote, carbohydrate, vitamins, amino acids, botany, lipids, proteins, cell, cell wall, biotechnology, metabolities, enzymes, agriculture, horticulture, agronomy, bryology, plaleobotany, phytochemistry, enthnobotany, anatomy, ecology, plant breeding, ecology, genetics, chlorophyll, chloroplast, gymnosperms, sporophytes, spores, seed, pollination, pollen, agriculture, horticulture, taxanomy, fungi, molecular biology, biochemistry, bioinfomatics, microbiology, fertilizers, insecticides, pesticides, herbicides, plant growth regulators, medicinal plants, herbal medicines, chemistry, cytogenetics, bryology, ethnobotany, plant pathology, methodolgy, research institutes, scientific journals, companies, farmer, scientists, plant nutrition
Select Language:
 
 
 
 
Main Menu
Please click the main subject to get the list of sub-categories
 
Services offered
 
 
 
 
  Section: Biotechnology Methods » Molecular Biology
 
 
Please share with your friends:  
 
 

Isolation of Cotton Genomic DNA from Leaf Tissue

 
     
 
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

Collection and Storage of Cotton Leaf Tissue
  1. Harvest cotton tissue of interest. Separate the different leaf sample in a plastic bag.
  2. Chill the samples on ice for transport back to the laboratory.
  3. Remove the midribs, if desired, then put the each sample in a 50-mL polypropylene tube (PPT).
  4. Store the samples at –70°C until freeze-drying them.
  5. Freeze-dry (See Lyophilization below) the sample for 2 to 4 days.
  6. Store the freeze-dried leaf tissue in a 50-mL PPT tightly-capped at –20°C, or in a desiccator at room temperature.
Lyophilization of Cotton Leaves
  1. Freeze the cotton leaf sample at –70°C in a deep freezer.
  2. Transport the sample in an ice chest.
  3. Check to see that the drain plug is closed (left side of condenser) and be sure that the (break) switch is off and the ballast open.
  4. Turn on the bottom cool switch of the condenser.
  5. Turn on the top (pump, cool, control) switches of the chamber and set “shelf temperature control” to –40°C or –50°C. Then, wait for the chamber temperature to drop to the set level (it usually takes 1 to 3 hours).
  6. Turn on the bottom (pump) switch.
  7. Place the frozen samples on trays. Arrange in even layers. Placing a second layer of tubes in the bottom 2 trays is OK. It may be fun to place the probes into samples to watch the changing temperature.
  8. Close the chamber and wait for 1 to 2 minutes until the samples and chamber temperature equalize. During this time, ice crystals will coat metal parts of the chamber.
  9. Turn on the gauge switch, close the ballast, and wait for the vacuum to reach <100 mT (about 5 to 10 min). In the meantime, condenser temperature should be at least – 40°C.
  10. Set “shelf temperature control” to –20°C and let run at least 3 hrs, or overnight.
  11. Check the condenser periodically to be sure that the coil has not collected enough ice to lug it up (rarely a problem on a fresh run).
  12. After the run is complete, turn off the (gauge) switch and open the ballast.
  13. Turn on (break) and turn off vacuum (pump) switches simultaneously.
  14. Remove the samples and turn off top (pump, cool, control) switches.
  15. Turn off (break) switch.
  16. Close the cap of samples tightly immediately.
  17. Place samples in a dry, airtight environment as soon as possible and store out of direct sunlight.
Procedure
  1. Use fresh BEST or lyophilize the leaf tissue.
  2. Grind about 5 g of leaf tissue with mortar and pestle in liquid N2, transfer the powder into a 50-mL polypropylene tube (50 mL), and store at –20°C.
    1. Add 25 mL of ice-cold extraction buffer, mix well, and put on ice until centrifugation at 3750 rpm for 20 minutes (4°C); remove supernatant (use a swinging bucket rotor).
    2. Add 10 mL lysis buffer and vortex to resuspend pellet; incubate in 65°C water bath for 30 minutes. Mix the tubes periodically by stirring or rocking the tubes every 10 minutes.
    3. Add 12 mL of chloroform:octanol (24:1) and mix gently, inverting the tube, until an emulsion forms.
    4. Centrifuge at 3750 rpm for 20 minutes (15°C). Transfer the upper phase to a new 50-mL tube through Miracloth.
    5. Add an equal volume of cold isopropanol, mix gently, and let it sit at room temperature for more than 1 hour.
    6. Centrifuge at 2500 rpm for 10 minutes at room temperature. Decant supernatant, and let tubes air dry. Then add 10 mL cold 76% ethanol/ 0.2 M Na Acetate. Leave at 4°C for 1 hour or overnight. (Good stopping point).
    7. Centrifuge tubes (3750 rpm, 10 min). Carefully discard supernatant, and rinse the pellet with cold 70% EtOH. Let tubes dry upside down on paper towels (keep track of tube labels!).
    8. Let pellets dissolve into 3 mL TE buffer. Vortex at speed 5, and incubate at 65°C for about 1 hour.
    9. Centrifuge at 3750 rpm for 10 minutes (15°C). Transfer supernatant to a new 15-mL tube. Add RNAse to a concentration of 20 µg/mL, mix gently, and incubate at 37°C for 15 minutes.
    10. Add 0.1 volume of 3M Na Acetate and 2 volumes of 95% ethanol for DNA precipitation. Place at –20°C for 1–12 hours (overnight).
    11. Spool/scoop out the DNA on a 9" glass-hook pipette (new and unused) and let it air dry under the hood or vacuum. If DNA cannot be spooled, centrifuge at 2500 rpm for 10 min to form DNA into pellet. Once the pellet is dry, place the pellet into 0.5 mL TE buffer in a 1.5-mL microfuge tube for 30 minutes at 65°C.
    12. Determine both concentration and quality of the DNA with a spectrophotometer with a 40 µL/800 µL dilution (1/20), and by running digested and undigested DNA in a 1% agarose gel.

Extraction Buffer (pH 6.0)
  • 0.35 M glucose
  • 0.1 M Tris HCl (pH 8.0)
  • 5.0 mM Na-EDTA (pH 8.0)
  • 2% PVP 40000 MW
  • 0.1% DIECA, diethyldithiocarbamic acid (disodium salt)
  • 0.2% beta-mercaptoethanol or Na2S2O5(add when used)
Lysis Buffer
  • 0.1 M Tris HCl (pH 8.0)
  • 1.4 M NaCl
  • 20 mM Na EDTA (pH 8.0)
  • 2% CTAB
  • 2% PVP 40000 MW
    0.1% DIECA, diethyldithiocarbamic acid (disodium salt)
  • 0.2% beta-mercaptoethanol or Na2S2O5 (add when used).
 
     
 
 
     




     
 
Copyrights 2012 © Biocyclopedia.com | Disclaimer