To Isolate Plasmid Dna from a Bacteria Culture and Set up a Restriction Enzyme Digest for the Plasmid Dna That Had Been Isolated

Aim

Week 1

To isolate plasmid DNA from a bacteria culture and set up a restriction enzyme digest for the plasmid DNA that had been isolated.

Week 2

To analyze the digested DNA using agarose electrophoresis and to perform quantitation of plasmid DNA using UV absorbance and fluorescent assay.

Introduction

        The alkaline lysis preparation is the most commonly used method for isolating small amount of plasmid DNA. This method often uses sodium dodecyl sulfate (SDS) as a weak detergent to denature bacteria protein in the presence of NaOH, which acts to hydrolyze the chromosal and plasmid DNA. The mixture is then neutralized with potassium acetate where causing the covalently closed plasmid DNA to reanneal rapidly. The chromosomal DNA, bacterial proteins as well as SDS will easily precipitate and can be separated by centrifugation. In doing so the insoluble SDS traps the larger genomic DNA and removes it from the supernatant. Alcohol such as ethanol is used to concentrate the re-annealed plasmid DNA remained in the supernatan (Watson & Berry, 2003). Restriction Digestion is the process of cutting DNA molecules into smaller pieces with special enzymes called Restriction Endonucleases These special enzymes recognize specific sequences in the DNA molecule (for example GATATC) wherever that sequence occurs in the DNA.

        Mobility of a molecule under the influence of an electric field "is determined by its charge, its formula weight, the pore size of the matrix material and the strength of the electric field". As DNA and RNA have constant anionic charge/mass ratios (one phosphate for every nucleotide linkage), they travel through the gel at a constant speed in response to an electric current. The visualization of double stranded DNA in agarose gels is the fluorescent dye SYRB Green. The dyes will fluorescene when exposed to UV light (Silverstein, Silverstein & Nunn, 2002). Quantitation of nucleic acids is commonly performed to determine the average concentrations of DNA or RNA present in a mixture, as well as their purity. The techniques in quantitating plasmid DNA include UV absorbance and fluorescent assays.

Methods

Week 1

Isolation of plasmid DNA  

Bacteria containing the plasmid of interest is first grown, and then allowed to lyse with an alkaline lysis buffer consisting of a detergent sodium dodecyl sulfate (SDS) and a strong base sodium hydroxide. The detergent cleaves the phospholipid bilayer of  membrane and the alkali denatures the proteins which are involved in maintaining the structure of the cell membrane. Through a series of steps involving agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris is removed and the plasmid is isolated and purified and ready for quantitation.

Restriction enzyme digestion of plasmid DNA

In a microfuge tube, add H2O, restriction buffer and plasmid DNA. Mix them by gently tapping the tube with finger, follow by adding enzyme mixture and incubate for at 370 C for one hour and ready for electrophoresis analysis.

Analysis of digested DNA using agarose electrophoresis

Add loading dye to restriction digest then samples can be loaded into wells made in the gel during molding. When an electric field is applied, the DNA molecules are separated by the pores in the gel according to their size and shape and visualized on a UV light box.

Quantitation of plasmid DNA (UV absorbance)

Isolated and purified plasmid DNA is diluted with distilled water in a UV cuvette and measure absorbance at 260nm and 280nm using the spectrophotometer.

Quantitation of plasmid DNA (fluorescent assay)

Isolated and purified plasmid DNA is diluted with distilled water. Then, dilute again with SYBR Green fluorescent assay and mix well. Then pipette mixture to 96-well microplate and measure the fluorescence emission with PerkinElmer Victor microplate reader. Emission filter of 485nm and 535nm are used.

Results

Table 1. DNA concentrations for SYBR Green fluorescent assay.

Table 2. Plasmid DNA dilutions for SYBR Green fluorescent assay.

Table 3