Transcript Plasmid Miniprep - California State University

Plasmid Miniprep
Broad and Long Term Objective
To characterize a single clone from an
Emiliania huxleyi cDNA library using
sequence analysis
Definitions
cDNA (complementary DNA)
DNA copy of a gene that lacks introns and therefore consists solely
of the coding sequence. Made by reverse transcription.
cDNA Library
Collection of genes in their cDNA form, lacking introns
cDNA Library Construction
Laboratory Objective
To isolate plasmid containing a cDNA
insert from Emiliania huxleyi, to use as a
template in a DNA sequencing reaction
Research Plan
Preparation of Competent Cells/Bacterial Transformation
Growth of Transformant and Plasmid MiniPrep
DNA Sequencing
Sequence Analysis
BLASTN/BLASTX /ORF Finder/Clustal W
Today’s Laboratory Objectives
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To isolate high quality plasmid DNA that can be used as
template for DNA sequencing
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To quantify and determine the purity of the isolated
plasmid DNA
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To determine the size of the plasmid DNA and its insert
Map of Parent Vector pMAB58
SnaBI
BspEI
SwaI
PpuMI
XbaI
Bsu36I
ARS4/Cen6
TRP1
7000
DraIII
Amp
AhdI
1000
F1
6000
BamHI
pT7
7577 bps
Ter ADH
attB2
pMAB58
2000
ori
5000
ccdB
3000
SacII
++ AatII
4000
attB1
BsrGI
MluI
AD
NLS
NotI
pADH
EcoRI
BsaBI
SmaI
BsrGI
BsmI
XbaI
EcoRI
PacI
MluI
BsrGI
SexAI
BseRI
Eco47III
RsrII
NspV
BsaBI
Asp718I
KpnI
ApaI
Bsp120I
StyI
BsmI
AlwNI
Theoretical Basis of the
Alkaline Lysis Plasmid Miniprep
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Lyse Cells
Separate nucleic acids from other
cellular macromolecules
Concentrate nucleic acids
Separate RNA from DNA
Alkaline/SDS Cell Lysis
SDS: anionic detergent causes cell lysis
Sodium hydroxide: base, denatures DNA
Selective Precipitation
Potassium Acetate
Ice
Centrifugation
Separation of Nucleic Acids
RNase
PEG Precipitation
For Cleaner DNA Precipitate w/ Polyethylene Glycol and NaCl
Theoretical Basis of UV
Spectrophotometry
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A UV spectophotometer measures the amount of light particular
molecules absorb (Proteins at A280; Nucleic Acids at A260)
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Lambert-Beer law describes the relationship between absorptivity
coefficient and concentration and is given by the following equation:
A=εbc
Where:
b= light path length
c=concentration of substance
ε=extinction coefficient
For DNA the extinction coefficient, ε= 1/50 ug/ml
Theoretical Basis of UV
Spectrophotometry
To Quantify your DNA sample:
A260 x Dilution Factor x 50 ug/ml= concentration of
nucleic acids 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
Theoretical Basis of Agarose Gel
Electrophoresis
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Agarose is a polysaccharide from marine alage that is
used in a matrix to separate DNA molecules
Because DNA ia a (-) charged molecule when subjected
to an electric current it will migrate towards a (+) pole
Pouring an Agarose Gel
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Sizing a Piece of DNA
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The distance the DNA migrates is dependent upon
the size of the DNA molecule
the secondary structure of the DNA
the degree of crosslinking in the gel matrix
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Size of DNA molecule can be determined by using
standards of known molecular weight
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a standard curve is made by plotting the molecular weights of the
standards and the distance each fragment has migrated from the
measuring the distance the unknown fragment migrated from the
well
substituting the distance the unknown migrated into the equation of
the line of best fit, and solving for Y (the molecular wt)