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University of Ottawa

LAB 5 Nucleic AcidsPurposeIn this laboratory four experiments will be performed In experiment one the DNA of EColi will be extracted For experiment two a stock solution of E coli DNA from a commercial source will be analyzed with melting curves Melting curves involve the analysis of the transformation of DNA from double stranded DNA to single stranded DNA with the addition of heat The type of DNA is identified through absorbance The melting curves will show the effects of salt and DMF on the stability of the commercial E Coli DNA The same melting technique will compare the homogeneity and the integrity of an inlab prepared DNA with a Commercial source of E Coli DNA The unknown plasma used in this laboratory was unknown plasma 42 Experiments three and four introduce the techniques of restriction digestion and DNA electrophoresis on agaroseR1Ultraviolet spectrophotometry is used to quantitate the amount of DNA and its purity Proteins absorb UV light with a maximum absorbance at 280nm due to the presence of the aromatic amino acids tryptophan Trp and tyrosine Tyr UV light with a maximum absorbance at 260nm is due to characteristics of the bases purines and pyrimidines along with nucleic acids both DNA and RNA An AAratio shows the relative amount of the different nucleotides in a 260280 specific DNA sample An AAratio between 18 and 19 shows a pure double stranded DNA 260280 sample higher ratios are due to RNA contamination Lower ratios of the AAfunction are 260280 caused by contamination by proteins An indicator of protein contamination is shown by the ration AA Nucleic acids have an absorbance minimum at 234 nm protein causes an 234260increase of the absorbance at this wavelength A AA ratio higher than 050 shows protein 234260contaminationAARatio Calculation260280 A0542260A0312280AA0542031217372260280Therefor the AA ratio was 17372 Since a ratio between 18 and 19 shows pure double 260280stranded DNA there was contamination of the sample from proteins This could have occurred when the upper aqueous phase was removed after centrifuging the sample If part of the lower phase was transferred to the 15ml falcon tube the AA ratio would be less than 1819 260280optimal range due to protein contaminationAA Ratio Calculation234260A0542260A0234234AA023405420432234260Therefor the AA ratio was 0432 Since the ratiois less than 05the protein contamination 234260isnt as detrimental as previously found with the AA The AA is a better indicator of 260234260280protein contamination because nucleic acids have a absorbance minimum at 234nm protein causes an increase of the absorbance at this wavelength With a AA value of 0432 there is 234260some protein contamination but it is not detrimental to the sample because the value is not greater than 05R2Figure 1 Commercial E coli DNA with no additives The following graph represents the absorbance and the derivative of the commercial DNA sample The point at which the
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