Molecular Biology Working with DNA

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

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

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

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

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

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

Plasmid DNA isolation by alkaline lysis (E.coli )

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

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??

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

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

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 +

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

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

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) 10 000 1 8 000 1.3 6 000 1.6 5 000 1.9 4 000 2.2 3 000 2.7 Etc

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

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

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 103.55 = 3548 bp

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

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?