Rec. DNA Lab Wednesday, Jan 24, 2007

Isolation of Chromosomal DNA from Photobacterium leognathi

Chromosomal DNA Isolation A lysozyme/SDS lysis procedure

1. Grow culture

2. Lyse cells

3. Remove unwanted cellular components

4. Precipitate DNA and dissolve in appropriate buffer

• Some important reagents:– Lysozyme– EDTA– SDS– Phenol/Chloroform– Proteinase K– Alcohol/Sodium

Acetate

Chromosomal DNA IsolationImportant techniques

• Phenol/Chloroform Extractions– Both reagents dissociate protein from nucleic

acids– Also remove lipids and some

polysaccharides– Aqueous layer (top) will contain DNA– Organic layer (bottom)– Proteins will be found at the interface

between the two layers – removal along with the aqueous layer is undesirable

Chromosomal DNA IsolationImportant techniques

• Alcohol precipitations– Allow the removal of nucleic acids from

solution as an insoluble pellet– A monovalent cation (Sodium Acetate, etc) is

required – 2 volumes of cold ethanol (or 0.7 vols

isopropanol) are then added– The precipitate is collected by centrifugation

DNA IsolationProcedure

• We will step through the outlined protocol as a class

Rec. DNA Lab Thursday, Jan 26, 2007

Spectrophotometric Analysis of DNA

Spectrophotometric Analysis of DNA

• UV spectrophotometry can be used to determine the quantity of DNA in a sample

• Nucleic acids strongly absorb UV light at 260 nm

• A solution of pure, double-stranded DNA at 50 μg/ml has an A260 of 1.0

• Absorbance at other wavelengths is useful:– 320 nm – particulate

contamination– 280 nm - RNA

contamination• A260:A280 ratio should be

1.8 – 1.9• RNA increases ratio • But protein decreases it

– 234 nm – protein contamination

• A234:A260 > 0.5 indicates contamination

Spectrophotometric Analysis of DNA

• Each group:– Spectrophotometric Analysis of the chromosomal

DNA isolated today– Spectrophotometric Analysis of the plasmid stock

prepared previously

• Determine the concentration of each sample• Determine if your samples are contaminated

using the relevant absorbance ratios