Plasmid Miniprep - PowerPoint PPT Presentation

1 / 16
About This Presentation
Title:

Plasmid Miniprep

Description:

Map of Parent Vector pMAB58. Theoretical Basis of the. Alkaline Lysis ... light particular molecules absorb (Proteins at 280 nm; Nucleic acids at 260 nm) ... – PowerPoint PPT presentation

Number of Views:215
Avg rating:3.0/5.0
Slides: 17
Provided by: drread
Category:

less

Transcript and Presenter's Notes

Title: Plasmid Miniprep


1
Plasmid Miniprep
2
Broad and Long Term Objective
  • To characterize a single clone from an
  • Emiliania huxleyi cDNA library using
  • sequence analysis

3
Research Plan
Preparation of Competent Cells/Bacterial
Transformation
Growth of Transformant and Plasmid MiniPrep
DNA Sequencing
Sequence Analysis
4
Todays Laboratory Objectives
  • To isolate high quality plasmid DNA that can be
    used as
  • template for DNA sequencing
  • To quantify and determine the purity of the
    isolated
  • plasmid DNA
  • To determine the size of the plasmid DNA and its
    insert

5
Map of Parent Vector pMAB58
6
Theoretical Basis of the Alkaline Lysis Plasmid
Miniprep
  • 1. Lyse Cells
  • 2. Separate nucleic acids from other cellular
    macromolecules
  • 3. Concentrate nucleic acids
  • 4. Separate RNA from DNA

7
1. Lyse Cells
  • Alkaline/SDS Cell Lysis
  • SDS anionic detergent that solubulizes
  • membranes and denatures proteins
  • Sodium hydroxide pH to 12.0,
  • denaturation of DNA

8
2. Separation of nucleic acids from other
cellular macromolecules
NaOH
K-acetate
protein
9
3. Concentration of Nucleic Acids
Precipitation 1 Isopropanol cations (Na)
Precipitation 2 PEG cations (Na) (increased
purity for sequencing)
10
4. Separation of RNA from DNA
RNaseA
11
Theoretical Basis of UV Spectrophotometry
  • A UV spectophotometer measures the amount of
    light particular molecules absorb (Proteins at
    280 nm Nucleic acids at 260 nm)
  • Lambert-Beer law describes the relationship
    between absorbtivity coefficient and
    concentration and is given by the following
    equation
  • Aebc
  • Where b light path length (cm)
  • cconcentration of substance
  • (ug/ml)
  • eextinction coefficient
  • For DNA the extinction
  • coefficient, e _____1_____
  • 50 ug/ml cm

12
Theoretical Basis of UV Spectrophotometry
  • To Quantify your DNA sample
  • Abs 260nm x Dilution Factor x 50 ug/ml
    concentration of DNA in a sample using a 1 cm
    pathlength
  • To estimate the purity of your sample
  • A260/A280 ratio of nucleic acids/protein
  • A260/A280 1.6-1.8 is optimal for DNA

13
Theoretical basis of restriction endonuclease
digestion
  • Type II restriction endonucleases cleave
  • DNA at specific, usually palindromic,
  • sequences

14
Theoretical Basis of Agarose Gel Electrophoresis
  • Agarose is a polysaccharide from marine algae
    that forms a matrix allowing separation of DNA
    molecules
  • Because DNA is a (-) charged molecule, when
    subjected to an electric current it will migrate
    towards a () pole
  • Separation based upon DNA size and secondary
    structure

15
Pouring an Agarose Gel
3
1
2
4
5
6
7
8
9
16
Sizing a Piece of DNA
  • Size of DNA molecule can be determined by using
    standards of known size
  • 1. A standard curve is made by plotting the
    size (in
  • bp) of the standards (Y-axis) against the
    distance
  • each fragment has migrated from the well
    (X-
  • axis)
  • 2. Measure the distance the unknown fragment
    migrated from the well
  • 3. Substituting the distance the unknown
    migrated
  • into the equation of the line of best
    fit, and
  • solving for Y (the size)
Write a Comment
User Comments (0)
About PowerShow.com