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  Section: Biotechnology Methods » Molecular Biology
 
 
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Restriction Digestion Methods—Restriction Enzyme Digests

 
     
 
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

The unit definition of restriction enzyme activity is based on the amount of enzyme required to cut bacteriophage lambda DNA to completion in 1 hour’s time. Assays developed by the manufacturer of the enzymes are most likely
done using highly purified DNA (i.e., plasmids or lambda phage DNA) as substrate, and assay conditions that produce the best results with their particular preparation. Often, the laboratory conditions are not as ideal, and a slight excess of enzyme or a longer incubation period is used to help ensure complete digestion.

There are many universal enzyme buffers that will work with a variety of enzymes, but often they do not meet the most efficient requirements for any one enzyme. Because the universal buffers are not as efficient, we do not recommend their routine use in the lab (especially for complex genomic DNAs; digests of complex genomic DNAs are usually at high DNA concentrations, which will inhibit the enzyme; also, the relatively crude DNA preps may contain inhibitors that require more units per enzyme and longer incubation times for complete digestion). It is always best to use the manufacturer’s recommended assay conditions for restricted digestion. Some manufacturers have cloned enzymes that have very different requirements from other versions of the same enzyme, so check before using. Enzyme concentrations are always provided on the side of the enzyme tube. Restriction enzymes used in the lab are always stored at –20°;C (in a glycerol base), and should be kept as close to –20°;C as possible to extend the life of the enzyme. When setting up digests, bring the reaction tube to the enzyme freezer in an ice bucket; remove the enzyme tube from the freezer and keep the tube on ice while working with the enzyme. Immediately return the tube to the freezer when finished. Use the pipetmen designated for restriction enzymes only, and always use a fresh pipetman tip when removing enzyme from the stock tube.

The lab has prepared enzyme buffer stocks. Check the requirements of the enzyme and use the appropriate buffer. Nearly all the manufacturers of enzyme now supply buffers with the enzyme, and these can be found in the enzyme freezer. Never make up a digest with the enzyme as more than 1/10 the final volume; the glycerol will inhibit at higher concentrations. Always set up a control digest when using an enzyme. Use a substrate DNA, which has known sites for the enzyme so that you can compare the control result to the expected pattern of fragments on an agarose gel. If preparing double enzyme digests, check the salt content of the buffers and use the enzyme with the lower salt requirements first, then adjust the reaction tube’s salt concentration and digest with the second enzyme. Impurities present in some human DNA preparations may inhibit restriction digestion. If you cannot obtain complete digestion after adding additional enzyme, set up another digest and add spermidine to a final concentration in the digest of 2 mm.

Restriction Enzyme Buffers



Stock Solutions for Restriction Enzyme Buffers

Always check the enzyme manufacturer’s recommended buffer conditions before setting up digests. Prepare the 10X buffers by combining all ingredients except the BSA. Filter sterilize, then add the sterile BSA. Prepare 1-mL aliquots, and store at the recommended temperatures. Buffers containing 2-ME can be stored in the refrigerator for a few weeks. If the mercaptoethanol is undetectible in refrigerated buffer, adding 6 mL of the 12M 2-ME stock per mL will restore the buffer
- DTT (dithiothreitol), 100 mM stock: Prepare in dH2O, filter sterilize and store at –20°;C.
- NaCl, 5 M stock: Prepare in dH2O, filter sterilize, and store at room temperature.
- KCl, 1 M stock: Prepare in dH2O, filter sterilize, and store at room temperature.
- Tris-HCl, pH 7.5-8.3, 1 M stock: Filter sterilize lab stock and store at room temperature.
- MgCl2., 1 M stock: Filter sterilize lab stock and store at room temperature.
- 2-mercaptoethanol (2-ME), 12 M stock: Store in the refrigerator. Pipette under the hood.
- BSA (bovine serum albumin), 50 mg/mL stock: Purchase from BRL, store at –20°;C.
  • Weigh out 0.25 g spermidine tetrahydrochloride.
  • Add double distilled H2O to bring volume to 10 mL.
  • Filter, sterilize, and store at 4°;C.

 
     
 
 
     




     
 
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