Plasmid MiniprepPlasmid Miniprep

Broad and Long Term ObjectiveBroad and Long Term Objective

To characterize a single clone from an To characterize a single clone from an Emiliania huxleyiEmiliania huxleyi cDNA library using cDNA library using sequence analysissequence analysis

DefinitionsDefinitions

cDNA (complementary DNA)cDNA (complementary DNA)

DNA copy of a gene that lacks introns and therefore consists solely DNA copy of a gene that lacks introns and therefore consists solely

of the coding sequence. Made by reverse transcriptionof the coding sequence. Made by reverse transcription..

cDNA LibrarycDNA Library

Collection of genes in their cDNA form, lacking intronsCollection of genes in their cDNA form, lacking introns

cDNA Library ConstructioncDNA Library Construction

Laboratory ObjectiveLaboratory Objective

To isolate plasmid containing a cDNA To isolate plasmid containing a cDNA insert from insert from Emiliania huxleyi,Emiliania huxleyi, to use as a to use as a template in a DNA sequencing reactiontemplate in a DNA sequencing reaction

Research PlanResearch Plan

Preparation of Competent Cells/Bacterial Transformation

Growth of Transformant and Plasmid MiniPrep

DNA Sequencing

BLASTN/BLASTX /ORF Finder/Clustal W

Sequence Analysis

Today’s Laboratory ObjectivesToday’s Laboratory Objectives

To isolate high quality plasmid DNA that can be used as To isolate high quality plasmid DNA that can be used as template for DNA sequencingtemplate for DNA sequencing

To quantify and determine the purity of the isolated To quantify and determine the purity of the isolated plasmid DNAplasmid DNA

To determine the size of the plasmid DNA and its insertTo determine the size of the plasmid DNA and its insert

Map of Parent Vector pMAB58 Map of Parent Vector pMAB58

pMAB58

7577 bps

1000

2000

3000

4000

5000

6000

7000

SwaIPpuMI

AhdI

AlwNI

Asp718IKpnIApaIBsp120I

StyIBsmI

PacIBsrGI

BsaBINspV

RsrIIEco47IIIBseRI

SexAIMluI

EcoRIXbaI

BsmIBsrGISmaI

BsaBIEcoRI

NotIMluI

BsrGIAatII++

SacII

BamHI

DraIII

Bsu36IXbaI

BspEISnaBI

ARS4/Cen6

Amp

ori

pADHNLS

ADattB1

ccdB

attB2

Ter ADH

pT7

F1

TRP1

Theoretical Basis of the Theoretical Basis of the Alkaline Lysis Plasmid MiniprepAlkaline Lysis Plasmid Miniprep

1.1. Lyse CellsLyse Cells

2.2. Separate nucleic acids from other Separate nucleic acids from other cellular macromolecules cellular macromolecules

3.3. Concentrate nucleic acidsConcentrate nucleic acids

4.4. Separate RNA from DNASeparate RNA from DNA

Alkaline/SDS Cell Lysis Alkaline/SDS Cell Lysis

SDS: anionic detergent causes cell lysisSDS: anionic detergent causes cell lysis

Sodium hydroxide: base, denatures DNASodium hydroxide: base, denatures DNA

Selective PrecipitationSelective Precipitation

Potassium AcetatePotassium Acetate

IceIce

CentrifugationCentrifugation

Separation of Nucleic AcidsSeparation of Nucleic Acids

RNase RNase

PEG PrecipitationPEG Precipitation

For Cleaner DNA Precipitate w/ Polyethylene Glycol and NaClFor Cleaner DNA Precipitate w/ Polyethylene Glycol and NaCl

Theoretical Basis of UV Theoretical Basis of UV SpectrophotometrySpectrophotometry

A UV spectophotometer measures the amount of light particular A UV spectophotometer measures the amount of light particular molecules absorb (Proteins at A280; Nucleic Acids at A260)molecules absorb (Proteins at A280; Nucleic Acids at A260)

Lambert-Beer law describes the relationship between absorptivity Lambert-Beer law describes the relationship between absorptivity coefficient and concentration and is given by the following equation:coefficient and concentration and is given by the following equation:

A=εA=εbcbc

Where: Where: bb= light path length= light path lengthcc=concentration of substance=concentration of substanceε=extinction coefficientε=extinction coefficient

For DNA the extinction coefficient, ε= 1/50 ug/mlFor DNA the extinction coefficient, ε= 1/50 ug/ml

Theoretical Basis of UV Theoretical Basis of UV SpectrophotometrySpectrophotometry

To Quantify your DNA sampleTo Quantify your DNA sample:: A260 x Dilution Factor x 50 ug/ml= A260 x Dilution Factor x 50 ug/ml=

concentration of concentration of nucleic acids in a sample using a nucleic acids in a sample using a 1 cm pathlength1 cm pathlength

To estimate the purity of your sampleTo estimate the purity of your sample::A260/A280= ratio of nucleic acids/proteinA260/A280= ratio of nucleic acids/proteinA260/A280= 1.6-1.8 is optimal for DNAA260/A280= 1.6-1.8 is optimal for DNA

Theoretical Basis of Agarose Gel Theoretical Basis of Agarose Gel ElectrophoresisElectrophoresis

Agarose is a polysaccharide from marine alage that is Agarose is a polysaccharide from marine alage that is used in a matrix to separate DNA moleculesused in a matrix to separate DNA molecules

Because DNA ia a (-) charged molecule when subjected Because DNA ia a (-) charged molecule when subjected to an electric current it will migrate towards a (+) poleto an electric current it will migrate towards a (+) pole

Pouring an Agarose GelPouring an Agarose Gel

1 2 3

4 5 6

7 8 9

Sizing a Piece of DNASizing a Piece of DNA The distance the DNA migrates is dependent uponThe distance the DNA migrates is dependent upon

the size of the DNA moleculethe size of the DNA molecule

the secondary structure of the DNAthe secondary structure of the DNAthe degree of crosslinking in the gel matrixthe degree of crosslinking in the gel matrix

Size of DNA molecule can be determined by using Size of DNA molecule can be determined by using standards of known molecular weightstandards of known molecular weight

1. a standard curve is made by plotting the molecular weights of the1. a standard curve is made by plotting the molecular weights of the standards and the distance each fragment has migrated from the standards and the distance each fragment has migrated from the

2. measuring the distance the unknown fragment migrated from the2. measuring the distance the unknown fragment migrated from the wellwell 3. substituting the distance the unknown migrated into the equation of3. substituting the distance the unknown migrated into the equation of the line of best fit, and solving for Y (the molecular wt)the line of best fit, and solving for Y (the molecular wt)