OVERVIEW PLASMID

• Plasmids


  Vehicles of recombinant DNA
• Bacterial cell

  • Non-chromosomal DNA
  • Replication: independent of the chromosome
  • Many copies per cell
  • Easy to isolate
  • Easy to manipulate

  
• Plasmid DNA isolation

  1. Separate plasmid DNA from the chromosomal DNA in the bacterial cell

  2. Separate from the polysaccharides, lipids and proteins that constitute the cell
• Miniprep

  Method for isolating smaller amounts of plasmid

  
• Maxiprep


  Method for isolating larger amounts of plasmid

  
• SDS-alkaline denaturation


  Principle used in manual plasmid isolation method

  • Overnight isolation
  • Resuspension sol. = buffer cont. Tris (main buff. subst), Glucose, EDTA
  • Alkaline lysis = cont. NaOH & SDS
  • Potassium acetate / acetic acid solution : A-Acetic acid : Neutralizes NaOH, Potassium acetate: Converts soluble SDS to insoluble PDS
• Rapid boiling method

  Another principle used for plasmid isolation
• DNA is very sensitive to mechanical stress, therefore shearing forces caused by mixing/vortexing or fast pipetting must be avoided as soon as cell lysis occurs
• All mixing steps during and after cell lysis should be performed carefully by inverting the tubes several times (4-5 times)
• Gloves should be worn in order to prevent contamination, new tubes and sterile tips should be used
• For long-term storage, plasmid DNA should be frozen in aliquots of storage TE buffer. Repeated thawing and freezing of DNA should be avoided
• SDS-alkaline denaturation method: Overnight Cult. Suspension


  1. Pick a single colony and inoculate in LB with ampicillin

  2. Incubate overnight at 37°C

  3. Centrifuge 1.5 mL of broth containing bacterial cells

  4. Discard supernatant
• Resuspension solution (soln1)

  
  

  Glucose : Give osmotic shock, lead to rupture of c.w & membrane

  Tris: main buffer substance, to maintain the pH of the buffer at a stable point, Interact w lipopolysacc. , then destabilize c.m

  EDTA: chelates divalent metals ( Mg2+ / Ca2+) & inhibits Dnases activity of cellular enzymes.
• Alkaline lysis solution (soln2)


  Contains:

  SDS = Dissolves membranes (disintegrates the lipid structure of c.m) , Binds to and denatures proteins

  NaOH = denatures both plasmid and chrom. DNA to SS.

  
  

  Chromosomal DNA separate completely into SS but plasmid not fully separate and remain entangled after denaturation
• Solution 3:Potassium acetate/acetic acid solution

  
  

  • Acetic acid = to neutralize NaOH, allow plasmid DNA to renature while chromosomal DNA remains denatured
  • Potassium acetate: Converts soluble SDS to insoluble PDS. Potassium salt of SDS is insoluble, so protein & detergent precipitate and aggregate, which entraped high-molecular-weight DNA.
• Separate plasmid DNA from contaminants

  Centrifugation separates supernatant containing plasmid DNA from pellet containing chromosomal DNA, proteins, lipids

  
• PureYield Plasmid Miniprep System

  Isolates high-quality plasmid DNA using silica-membrane column purification
• Plasmid yield will vary depending on culture volume, plasmid copy number, culture medium, and bacterial strain
• For best results, use plasmids that are 10,000bp or less

  
• Centrifugation Protocol for PureYield Miniprep

  1. Transfer bacterial culture to tube

  2. Add Cell Lysis Buffer, mix by inverting

  3. Add cold Neutralization Solution, mix thoroughly

  4. Centrifuge to pellet debris

  5. Transfer supernatant to minicolumn

  6. Centrifuge minicolumn

  7. Wash minicolumn with Endotoxin Removal Wash and Column Wash Solution

  8. Elute plasmid DNA from minicolumn
• Methods plasmid isolation

  • Depends on amount plasmid isolated = Miniprep & Maxiprep
  • Depends on principle = SDS-alkaline denat. & Rapid boiling
• PureYield™ Plasmid Miniprep System Protocol :

  1. Centrifugation Protocol.
  2. Vacuum Protocol.
  3. Alternative Protocol for Larger Culture Volumes.
• Component of kit: (30–37 °C )

  • Cell Lysis Buffer (CLC).

  • Neutralization Solution (NSC) -store at 4-8 °C)

  • Endotoxin Removal Wash (ERB).

  • Column Wash Solution (CWC).

  • Elution Buffer (EBB). Tris-HCl (pH 8.5), EDTA.

  •PureYield™ Minicolumns

  • PureYield™ Collection Tubes
• Sol.3 : Separate plasmid DNA from contaminants centrifugation results

  Supernatant cont.: Plasmid DNA, Some cellular constituents

  Sediment cont.: PDS, Lipids, Proteins, Chromosomal DNA

  • The soluble plasmid DNA (supernatant) is ready to be further purified.