Digestion of extracted plasmid

/Digestion of extracted plasmid
Digestion of extracted plasmid2018-02-26T11:51:31+00:00

Digestion of extracted plasmid

Digestion of extracted plasmid

Introduction

Restriction endonucleases are enzymes that recognize specific nucleotide sequences in double-stranded DNA and are a major tool of the biotechnologist. For prokaryotic cells, they function in nature as restriction-modification systems and will cleave foreign DNA that enters the bacterial cell (e.g., bacteriophage) but will not cleave host DNA that has been “protected” or modified by methylation. Most restriction endonucleases will reproducibly cleave DNA at a precise point within a recognition sequence. Generally, different enzymes will recognize different sequences that are four to six nucleotides long. This precision is essential for molecular cloning techniques, such as isolating genes from genomic DNA or inserting foreign DNA into a plasmid. Many enzymes make reproducible staggered cuts with respect to the dyad axis of symmetry of their recognition sequence. Cleavages of this type will yield DNA fragments with 3′ and 5′ overhangs or “sticky ends” that can base pair with DNA fragments generated by the same enzyme and thus form recombinant molecules.

Other enzymes will cut the recognition site at the axis of symmetry to yield blunt-ended cleavage products. These can be ligated to other blunt-ended fragments irrespective of the restriction enzyme used. The second technique introduced in this exercise is agarose gel electrophoresis. When agarose is melted and then cooled in an aqueous solution, it forms a gel by hydrogen bonding. The population of DNA fragments generated by restriction enzymes will move through an agarose gel under the influence of an electric field, where negatively charged DNA molecules will be drawn to the anodes. Their rate of movement is based almost entirely on size, with the largest molecules having the lowest mobility’s. The concentration of agarose in the gel determines pore size, and a DNA fragment having a particular size will migrate at different rates through gels of different concentrations. There is a linear relationship between the log of mobility and gel concentration over a certain range of fragment sizes, so a gel concentration must be chosen that will effectively separate the molecules in the DNA population. Gels of 0.8% (w/v) agarose are suitable for separating linear DNA molecules 0.5-10 kb in size. The log molecular weights of the known marker fragments, such as phage cut with the restriction nuclease HindIII, can be plotted against mobility.

The resulting calibration curve can be used to determine the molecular weights of unknown DNA fragments. As a quick measure, the molecular weight of an unknown fragment can be estimated by direct comparison by eye to the position of ~ fragments on the gel. The bands are visualized by ultraviolet (UV) light illumination after staining with the fluorescent dye, Ethidium bromide. (The appropriate quantity of DNA present in a band can be estimated as well by comparison of fluorescence intensities as in Exercise 5, Part C.) Purity and any degradation of the DNA can also be determined on agarose gels. Contamination with RNA can be detected as a broad smear running at low molecular weight. RNA can be removed by addition of RNase to restriction digests. Contamination with protein may result in partial inhibition of restriction enzyme activity or degradation of the DNA. Protein contaminants can be removed by phenol-chloroform extraction and subsequent ethanol precipitation. Excess salt in a plasmid preparation will often inhibit enzymatic activity as well. Salt can be removed by ethanol precipitation followed by washing of a DNA pellet with 70% (v/v) ethanol. The agarose gel will show if the digested plasmid yields the bands expected from known restriction sites on the plasmid map. If the DNA has been degraded, e.g., by contaminating DNases, the DNA will appear as a smear in the stained gel.

Requirements

Reagents

  1. Agarose
  2. DNA staining solution
  3. Electrophoresis buffer
  4. 6x Gel-loading buffer
  5. Lambda DNA
  6. DNA size standards
  7. EcoRI
  8. HindIII
  9. Assay Buffer

Equipments

  1. Horizontal electrophoresis buffer
  2. Power supply

Procedure

1. Place the vials containing restriction enzyme (EcoR I and Hind III) on ice.
2. Thaw the vials containing substrate (Lambda DNA) and assay buffer.
3. Prepare 2 different reaction mixtures using the following constituents.

Reaction 1 ( EcoR I digestion)

  • Lambda DNA 5µl
  • Assay buffer 2.5 µl
  • EcoR I  1µl
  • Mili Q water: 16.5 ul
    Total: 25 ul

Reaction 2 ( Hind III digestion)

  • Lambda DNA: 5 µl
  • Assay buffer: 2.5 µl
  • Hind III: 1 µl
  • Mili Q: 16.5 ul
    Total: 25 ul

4. Incubate the vial at 37°C for 1 hour.
5. Meanwhile, prepare a 1% agarose gel for electrophoresis.
6. After an hour add 5 μl of gel loading buffer to vials.
7. Load the digested samples, 10 μl of Control DNA and 10 μl of marker, note down the order of loading.
8. Electrophorese the samples at 50-100 V for 1-2 hours.

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