1. Laboratory Activity Nine
DNA Extraction &
Analysis

2. Purpose of Lab 09 Common Handling & Analyses:
Educational – provide experience in the handling and analysis of DNA.
Common Handling & Analyses:
Extraction
Purification
Quantification
Restriction digest
Amplification
Electrophoresis
Base sequencing
Cloning

3. DNA Extraction
Cell / tissue disruption to release DNA into extraction buffer.
Solubilize and remove membranes & lipids with detergents.
Remove protein contamination by protease treatment or phenol extraction.
Precipitate DNA
with alcohol.
Nucleolus
Heterochromatin
Euchromatin
An Interphase Nucleus
 30 nm 
(Nucleosomes)
10 nm
Phenol/chloroform extracts proteins into lower organic phase, some hang up at the interface.
Nucleosome has about two loops of DNA around histone core, and approx. 150 – 200 bp’s long.
Extraction buffer pH is 9.5 – alkaline to strip H+ from basic histone proteins (and AA’s – lysine & arginine), thus weakening the interaction between histone & DNA (which is negative).

4. Ethanol Precipitation
Slowly add ice-cold ethanol (so that it “floats” on top of the DNA extract).
Ethanol dilutes water, removing hydration shells of + ions, allowing DNA phosphate salts to form.
DNA salts are less soluble in alcohol solutions than aqueous solutions.
DNA precipitates at the water- ethanol interface.
Banana
Onion

5. Quantification of Nucleic Acids
Spectrophotometric, based on absorbance at 260 nm.
OD260 of 1.0 =
50 µg/mL double-stranded DNA*.
37 µg/mL single-stranded DNA.
40 µg/mL RNA.
OD260 can be problematic:
Protein absorbs strongly at 280 nm.
Protein is the most common contaminant in DNA preparations.
Purity / contamination can be estimated via OD260:OD280 ratio.
1.7 – 2.0 is good.
1.0 is bad (mostly protein). I
300
280
260
*µg/mL =
ABS260 x 50

6. UV-260/280 Interpretations ABS260/ABS280 % DNA % Protein 2.0 1.9 1.8
1.7
1.3
1.0
0.6
100 70 50 30 10 5 90 95

7. Restriction Digests Definition:
The enzymatic cleavage of higher MW DNA into smaller fragments with a “restriction endonuclease”.
Origin & Significance of Restriction Enzymes:
Defense enzymes produced by microbes.
Prevent (“restrict”) the takeover by foreign (viral) DNA.
Cut DNA at very specific recognition sites & patterns.
Applications in Molecular Biology:
Cut large cumbersome DNA molecules into smaller easily manageable fragments.
“Sticky ends” are extremely useful in cloning and recombination.

8. “EcoR1” (Restriction Enzyme no. 1 from E. coli)
“Sticky Ends”

9. “EcoR1” (Restriction Enzyme no. 1 from E. coli)
Recipient (host) DNA
Foreign (engineered) DNA
Recombinant DNA

10. Hind III Digest of Lambda DNA
Phage Lambda
 DNA
COS
Site
Lysis
genes
Head
Tail
DNA
Replication
Host
Integration
Hind III
Restriction enzyme from Haemophilus influenzae.
Phage 
Bacteriophage that infects E. coli.
One of the most studied viruses.
Contains “ DNA”.
Lambda DNA
48,502 bp long; circular or linear.
Complete sequence known.
Has 7 Hind III restriction sites.
H. influenzae was once considered to be the cause of influenza until 1933, when the viral role of the flu became apparent; Most strains of H. influenzae are opportunistic pathogens – they usually live in their host without causing disease, but cause problems only when other factors (such as a viral infection or reduced immune function) create an opportunity.
COS Site – is the Cohesive Site where lambda DNA naturally splits to “linearize” or “cyclize” itself; it’s about 12 overlapping nucleotides in length that results in “sticky ends”. Once inserted into a host cell, the COS sites base pair to cyclize the DNA. This helps prevent degradation via exonucleases. However, in the virus the DNA is linear.
Cos sequences are ~200 base pairs long and essential for packaging. 

11. Hind III Digest of Lambda DNA
7 Restriction sites
8 DNA fragments
Hind II Cuts at: A/AGCT T
T TCGA/A

12. Agarose Gel Electrophoresis
Theory & principles like PAGE.
Samples include:
MW markers (“DNA Ladder”).
Two concentrations of genomic DNA extract prepared from leaves.
Uncut  DNA.
 DNA cut with Hind III.
Caution: Agarose gels contain Ethidium Bromide.
Intercalating agent, mutagen.
Base pair insertions, deletions.
Fluoresces pink (under UV light) in the presence of DNA.
Caution: UV light causes cataracts & sunburns.

13. EZ - Vision DNA Stain
Active Ingredient = 4',6-diamidino-2-phenylindole (aka “DAPI”).
Binds to AT-rich regions of minor groove.
Fluoresces blue under UV light.
“Safer” than ethidium bromide.
Bovine pulmonary artery endothelial cells stained with DAPI

14. Interpretation of Results
Mainly . . .
Estimate the amount of DNA in 1 g of plant tissue.
For the DNA ladder components, make a graph of Rf vs. log10(no. of base pairs).
Use to estimate/confirm fragment sizes of Hind III digest of  DNA.
Speculate on differences in bp’s or absence of fragments.
Note – our agarose gels are normally 1% (not 0.7% as shown in above gel).

15. Questions or Comments