1. Molecular Biology
Working with DNA

2. DNA Genomic Extra-genomic Prokaryote vs. eukaryote Circular or linear
One or more chromosomes
Extra-genomic
Vectors
Plasmids

3. Vectors Vs Plasmids Vector:
DNA vehicle that allows the cloning, maintenance and amplification of a DNA sequence
Plasmids
Virus
Chromosomes
All plasmids are vectors
Not all vectors are plasmids

4. Plasmids
Small circular DNA molecules maintained and amplified in eukaryotic or prokaryotic cells
Amplification in bacteria
Used as vector for cloning or expression of DNA of interest

5. Characteristics of plasmid vectors
Restriction sites for cloning
Origin of replication (Ori)
Selection marker
Genes conferring resistance to antibiotics

6. DNA Isolation Goals Isolation of DNA of interest
Chromosomal or plasmid?
Eliminate other components
Chromosomal or plasmid DNA?
Proteins
RNA
Chemicals
Salts, detergents, etc.

7. DNA isolation (cont’d)
Cell lysis
Cell wall and membrane
Enzymatic
Chemical
Mechanical
Isolation of DNA of interest
Differential sedimentation
Chromatography
Removing other components

8. Plasmid DNA isolation by alkaline lysis (E.coli )

9. Solutions Used Sol. I – Resuspension buffer Sol. II – Lysis solution
Tris HCl – Buffer that protects nucleic acids
EDTA - Chelates Mg++, prevents nucleases from working
Sol. II – Lysis solution
NaOH - ^pH lyses cells, denatures DNA
SDS – Dissolves membranes, denatures and binds proteins

10. Solutions Used (Cont’d)
Sol. III- Potassium acetate
Renaturation of DNA
Precipitates SDS
Precipitates genomic DNA and proteins
Isopropanol / Ethanol
Precipitates nucleic acids (plasmid and ?)
Salts remain soluble
TE-RNase - Tris & EDTA again; RNase??

11. Quantification of DNA Determining Conc. of DNA
A260 of 1.0 = 50µg/mL or 50ng/µL
Determining Amount of DNA
1mL of a solution with an A260 of 1.0 contains 50µg DNA
1µL of a solution with an A260 of 1.0 contains 50ng DNA
Do not forget to account for the DILUTION FACTOR

12. Sample problems
You have a DNA sample from which a 1/50 dilution has an abs. 260 of 0.4. What is the original DNA concentration in µg/µL?
1 µg/µL
What volume of the undiluted sample would you need to have 10µg?
10 µL
You have a DNA solution at 20µg/mL. By what factor must this solution be diluted to obtain an abs. 260 of 0.01
40X

13. Agarose gel electrophoresis
Separation of single or double stranded nucleic acids according to their size and conformation
Separation of fragments between 100pb and 10 Kbp
Resolution of fragments ≥100pb

14. Undigested plasmid on a gel-
Undigested plasmid on a gel -
Undigested plasmids generate a pattern of bands
Migration is a function of size and conformation
Supercoiled
Relaxed
Multimers?
multimers
Relaxed
Supercoiled +

15. Migration of linear DNA-Digested plasmids
The migration speed is a function of the size
Smaller fragments migrate faster
The migration speed is inversely proportional to the log10 of the size

16. Migration of linear DNA-digested plasmids
Sample 1
Sample 2 -
1000 bp
850 bp
750 bp
600 bp
200 bp
100 bp +

17. Determining fragment sizes
Fingerprinting Standard Curve: Semi-log
Step 1
Generate a standard curve from molecular weight ladder
Determine migration distances corresponding to each size
Size (bp) Distance (mm)
Etc

18. Determining fragment sizes
Fingerprinting Standard Curve: Semi-log
Step 2
Enter data in Excel
Covert size to log

19. Determining fragment sizes
Fingerprinting Standard Curve: Semi-log
Step 3
Generate curve in Excel

20. Determining fragment sizes
Fingerprinting Standard Curve: Semi-log
Step 4
Determine unknown sizes according to migration distance
3.55
Take inverse log to obtain size
= 3548 bp

21. Visualization: Ethidium Bromide
Stain used to make nucleic acids visible
Fluorescent under UV
Binding is proportional to
The size
The quantity
The conformation

22. What can be determined from an electrophoresis on an agarose gel?
Is there DNA
How many conformations
How many fragments
The size of the fragments
Total size of nucleic acid molecules
The number of cuts
Linear?
Circular?