Lab #8 - Click to edit Master subtitle style LAB 8 DNA...

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Unformatted text preview: Click to edit Master subtitle style LAB 8 DNA Quantitation and Restriction digestion P H S 2 3 1- B I O M E D Objective • To determine the concentration and quality of pGLO plasmid DNA • To perform a restriction digest of pGLO plasmid • To perform a simulated restriction digests using computer software • To learn how to access genomic and proteomic databases and retrieve information Quantitation of DNA • Absorbance is proportional to the concentration • Absorbance is also known as Optical Density (OD) • The wavelength of light used depends on the target molecule • DNA, RNA and proteins are colorless • The wavelength of maximum absorbance of DNA and protein lie in the UV range • Spectrophotometer is used to measure absorbance Quantitation of DNA • DNA absorbs light maximally at 260nm – An OD260 of 1 corresponds to 50 μ g/ml of double stranded DNA (50 ng/ μ l of DNA) • RNA also absorbs light maximally at 260nm – An OD260 of 1 corresponds to 40 μ g/ml of single stranded RNA (40 ng/ μ l of RNA) • Remember unit conversion 1 mg = 1000 μ g = 1000000 ng 1 ml = 1000 μ l • Dilute DNA before measuring absorbance 5 μ l to 1 ml • Use 1 OD = 50 μ g/ml since you are quantifying DNA • Multiply by the dilution factor (200) to obtain concentration of the your undiluted DNA prep Quality of DNA DNA concentration for the crude prep procedure used last week is generally not more than 1 μ g/ μ l (1mg/ml) DNA preps prepared without RNAse enzyme contain RNA....
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This note was uploaded on 03/09/2011 for the course PHS 2301 taught by Professor Bohu during the Spring '11 term at St. John's.

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Lab #8 - Click to edit Master subtitle style LAB 8 DNA...

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