1. Rec. DNA Lab Wednesday, Jan 24, 2007 Isolation of Chromosomal DNA from Photobacterium leognathi

2. 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

3. Chromosomal DNA Isolation I mportant 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

4. Chromosomal DNA Isolation I mportant 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

5. DNA Isolation Procedure We will step through the outlined protocol as a class

6. Rec. DNA Lab Thursday, Jan 26, 2007 Spectrophotometric Analysis of DNA

7. 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 A 260 of 1.0 Absorbance at other wavelengths is useful: –320 nm – particulate contamination –280 nm - RNA contamination A 260 :A 280 ratio should be 1.8 – 1.9 RNA increases ratio But protein decreases it –234 nm – protein contamination A 234 :A 260 > 0.5 indicates contamination

8. 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