Friday, May 22, 2009

Cleave DNA Using Restriction Endonulease, A Bacterial Enzyme

In order to manipulate DNA you have to posses the ability to cleave DNA at specific sites by using bacterial enzyme, which is restriction endonulease. Restriction endonucleases are bacterial enzymes that cleave duplex DNA at specific target sequences with the production of defined fragments. The name of the enzyme (such as BamHl, EcoRl, AluI, and so on) tells us about the origin of the enzyme but does not give us any information about the specificity of cleavage. The recognition site for most of the commonly used enzymes is a short palindromic sequence, usually either 4, 5, or 6 bp in length, such as AGCT (for AZul),GAATTC (for EcoRl), and so on. Each enzyme cuts the palindrome at a particular site, and two different enzymes may have the same recognition sequence but cleave the DNA at different points within that sequence.

  1. 10X stock of the appropriate restriction enzyme buffer.
  2. DNA to be digested in either water or TE (10 mM Tris-HCl pH 8.3, 1 mM ethylenediaminetetraacetic acid [EDTA]).
  3. Bovine serum albumin (BSA) at a concentration of 1 mg/mL.

    BSA is routinely included in restriction digests to stabilize low protein concentrations and to protect against factors that cause denaturation.

  4. Sterile distilled water.

    Good-quality sterile distilled water should be used in restriction digests. Water should be free of ions and organic compounds, and must be detergent free.

  5. The correct enzyme for the digest.
  6. 5X Loading buffer: 50% (v/v) glycerol, 100 mM Na2EDTA, pH 8, 0.125% (w/v) bromophenol blue, 0.125% (w/v) xylene cyanol.
  7. 100 mM Spermidine.

    Digests of genomic DNA are dramatically improved by the inclusion of spermidine in the digest mixture to a final concentration of 1 mM since the polycationic spermidine binds negatively charged contaminants.
    Spermidine can cause precipitation of DNA at low temperatures, so it should not be added while the reaction is kept on ice.

  1. Thaw all solutions, with the exception of the enzyme, and then place on ice.
  2. Decide on a final volume for the digest, usually between 10 and 50 microliters, and then into a sterile Eppendorf tube, add 1/10 vol of reaction buffer, 1/10 vol BSA, between 0.5 and 1 micrograms of the DNA to be digested, and sterile distilled water to the final volume.

    The amount of DNA to be digested depends on subsequent steps. A reasonable amount for a plasmid digestion to confirm the presence of an insertion would be 500 ng-l microgram, depending on the size of the insert. The smaller the insert, the more DNA should be digested to enable visualization of the insert after agarose gel analysis.

  3. Take the restriction enzyme stock directly from the -20oC freezer, and remove the desired units of enzyme with a clean sterile pipet tip. Immediately add the enzyme to the reaction and mix.

    Stock restriction enzymes are very heat labile and so should be removed from -20oC storage for as short a time as possible and placed on ice.

  4. Incubate the tube at the correct temperature (see Note 12) for approx 1 h. Genomic DNA can be digested overnight.

    Note that the incubation temperature for the vast majority of restriction endonucleases is 37oC but that this is not true for all enzymes.

  5. An aliquot of the reaction (usually 1-2microliter) may be mixed with a 5X concentrated loading buffer and analyzed by gel electrophoresis.

Hopefully this method can help your work.


Reference : Number 14 on References