Restriction Enzyme Digest

Digesting E. huxleyi genomic DNA

BRAVO!!!
YOU DID IT!!!
You Isolated Genomic
DNA from E. huxleyi!!!
 Today’s Laboratory Objectives
 Determine the concentration, purity, and
integrity of the E. huxleyi genomic DNA

 Digest E. huxleyi genomic DNA

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

 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= 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

 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
1 2 3

4 5 6

7 8 9
 Sizing a Piece of DNA
 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

 Size of DNA molecule can be determined by using

standards of known molecular weight
1. a standard curve is made by plotting the molecular weights of the
standards and the distance each fragment has migrated from the
2. measuring 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 molecular wt)
Assessing the Integrity of DNA
High Quality Genomic DNA
>95% DNA will be of high molecular
weight, migrating as intact band near
the top of the gel

Very little evidence of smaller

fragments indicated by a smear of
many different sized DNA fragments
 Restriction Enzymes
 called "restriction enzymes“ because restrict host range for certain
bacteriophage
 bacterial" immune system": destroy any "non-self" DNA
 methylase recognizes same sequence in host DNA and protects it by
methylating it; restriction enzyme destroys
unprotected = non-self DNA (restriction/modification systems)
 Restriction Enzymes
 Hundreds of restriction enzymes have been
identified.
 Most recognize and cut palindromic
sequences
 Many leave staggered (sticky) ends
 by choosing correct enzymes can cut DNA
very precisely
 Important for molecular biologists because
restriction enzymes create unpaired "sticky
ends" which anneal with any
complementary sequence
Some Commonly Used
Restriction Enzymes

Eco RI 5'-G | AATTC

Eco RV 5'-GAT | ATC
Hin D III 5'-A | AGCTT
Sac I 5'-GAGCT | C
Sma I 5'-CCC | GGG
Xma I 5'-C | CCGGG
Bam HI I 5'-G | GATCC
Pst I I 5'-CTGCA | G
 Theoretical Basis
Using Restriction Enzymes
 The activity of restriction enzymes is dependent upon
precise environmental condtions:

PH
Temperature
Salt Concentration
Ions

 An Enzymatic Unit (u) is defined as the amount of enzyme

required to digest 1 ug of DNA under optimal conditions:

3-5 u/ug of genomic DNA

1 u/ug of plasmid DNA
Stocks typically at 10 u/ul
 Next Week

 Separate our restriction fragments using agarose gel

electrophoresis

 Southern Transfer- transfer denatured DNA from agarose gel to

a membrane on which it can be analyzed using a labelled
complementary DNA probe to PEPCK