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:  
 
 

Restriction Digestion of Plasmid, Cosmid, and Phage DNAs

 
     
 
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

Principle

The following is a generalized example of a restriction digest. Estimate the amount of DNA needed in your digest and scale up accordingly. To visualize a digest on an ethidium bromide-stained agarose gel, you will need to take the size of the fragments and the total size of the clone DNA into account (e.g., 10–50 ng of intact lambda-sized genomes (~50 kb) are easily seen on gels but if cut into small (~1 kb fragments), the relative proportion of the clone DNA in each fragment is ~1/50 and more DNA (500–1000 ng) should be loaded in order to see them.

If preparing a large number of digests at a time and the DNAs are at the same concentration, prepare a cocktail of the reaction mix then divide it among the tubes of DNA.


Time Required
1.5 hours


Procedure
A general rule of thumb is to use 1 mg clone DNA/ 10 mL in the final digest
reaction mix (recommendation 1 µg/20 µL).
Reaction Mix:

  1. Put the water and buffer into the tube first, then add the enzyme (avoid putting enzyme into water first, as it may start to break down). Put the DNA in last and mix by tapping the tube with your finger.
  2. Quickspin to remove bubbles (DNA will adhere to bubble surface and becomes inaccessible to the enzyme). Incubate at the recommended temperature for 1 hour.
  3. Stop the reaction by adding 2.5 µL 5 X Ficoll dye mix if the sample is to be loaded directly onto a gel; otherwise stop the reaction by placing it at –20°;C or add 0.5 µL of 0.5 M EDTA.

 
     
 
 
     




     
 
Copyrights 2012 © Biocyclopedia.com | Disclaimer