FMG calculations lab 2 Results: Table 1.

0 Absorbance values obtained by varying the volume of the stock DNA hydrolysate and reacting with diphenymamine and used to construct a calibration curve. Tube mL DNA-acid hydrolysate A600 DNA content(mg) 1 ---0.00 0.00 2 0.1 0.063 0.057 3 0.2 0.113 0.114 4 0.2 0.117 0.114 5 0.3 0.181 0.171 6 0.5 0.298 0.285 7 0.5 0.280 0.285 8 0.8 0.459 0.456

Sample calculations for determining the DNA using tube 3 as an example. The concentration of the stock DNA solution 1mg/mL The volume of the solution used was 2ml and the final volume of the solution was 3.5 ml conc is therefore 2mg/3.5mL = 0.57mg.mL-1 The mass of DNA in the solution was 0.2ml x = 0.114mg

Sample calculations to find the average absorbance of the Tube 3 and Tube 4 (0.113 + 0.117)/2 = 0.115

Graph 1.0 Calibration curve obtained buy diluting a stock contentration of DNA(1mg/ml) and diphenyamine reagent
0.5 0.45 0.4 Absorbance at 600nm 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 0.05 0.1 0.15 0.2 0.25 DNA mass (mg) 0.3 0.35 0.4 0.45 0.5 y = 1.014x

Sample calculation to determine the amount of DNA present in the Liver extract sample. Using the line of the equation y = 1.014x and the absorbance value of the 0.5ml liver extract solution of 0.077 x = 0.077/1.014 x = 0.76 mg of DNA was present in the 0.5ml sample To calculate the mg of DNA present in the 1ml sample x = 0.164/1.014 x = 0.162mg

Table 2.0 Absorbance values obtained by varying the volume of the stock RNA hydrolysate and reacting with orcinol and used to construct a calibration curve. Tube mL RNA-acid hydrolysate A660 RNA content(mg) 1 --0.00 0.00 2 0.1 0.149 0.013 3 0.2 0.324 0.027 4 0.2 0.325 0.027 5 0.3 0.517 0.040 6 0.4 0.634 0.052 7 0.4 0.640 0.052 8 0.5 0.786 0.067

Sample calculations for determining the RNA using tube 3 as an example. The concentration of the stock RNA solution 0.2mg/mL The volume of the solution used was added to the 1 ml of acid was 2 ml Therefore 0.4mg of RNA was added and the concentration was 0.4mg/3mL = 0.133mg.mL-1 The mass of RNA in the solution was 0.2ml x = 0.027mg

Graph 2.0 Calibration curve obtained by diluting a stock concentration of stock RNA(0.2mg/ml) and Orcinol reagent
0.9 0.8 Absorbance at 660nm 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 0.01 0.02 0.03 0.04 RNA mass (mg) 0.05 0.06 0.07 0.08 y = 12.112x

Table 3.0 Masses, volumes and absorption values obtained in the process of DNA and RNA extraction of tissues. Liver Mass of tissue(g) Final volume of homogenate(mL) DNA A600 RNA A660 --------0.5ml 1.0ml 0.2ml 0.4ml 12 126 0.077 0.164 0.155 0.310 Brain 9.2 97 0.057 0.111 0.042 0.094 Kidney 9.3 99 0.114 0.247 0.077 0.140

Table 4.0 amount of DNA and RNA present in 1mL each respective sample solution tested Tissue Liver Kidney Brain DNA(mg) 0.157 0.232 0.109 RNA(mg) 0.0635 0.0299 0.0182

Sample calculation for DNA determination using the Liver sample The equation of the line obtained from the calibration curve for the DNA is y = 1.014x For the 0.5ml liver sample DNA content and the 1ml Liver sample DNA content was = 0.076 mg of DNA = 0.162 mg of DNA

The average value for mg of DNA in 1.0ml of supernatant is calculated by First averaging the 0.5ml liver sample and the 1ml liver sample (0.076mg x 2) = 0.152mg = 0.157mg of DNA present in 1ml

Table 5.0 Total DNA content and RNA content of each organ Tissue Liver Kidney Brain Volume of supernatant obtained 4.3 4.6 4.6 Total DNA content(mg) 42.53 52.35 25.23 Total RNA content(mg) 86.01 34.07 20.273

Sample calculations for Liver in determining Total DNA content The total volume of the supernatant 4.3ml So the total amount of DNA present is given by 0.157mg x 4.3 = 0.675mg There was therefore 0.675mg of DNA in 2ml of the liver homogenate solution or = 0.338mg.ml-1

The total volume of the homogenate was 126ml so the total mg of DNA is calculated by 0.338mg.ml-1 x 126ml = 42.53mg There is a total of 42.53mg present in the 12g of liver = 3.54mg of DNA per gram of liver Table 6.0 Determined Concentrations of RNA and DNA in mg/g of each organ used in the experiment. Protocol Homogenates DNA RNA Concentration of Nucleic acid(mg/g) liver Kidney 3.54 5.66 7.17 3.664 Brain 2.71 2.20

Sample calculation using liver results to calculate the RNA content First find the concentration of RNA present in the diluted solution Using the equation of the line from Graph 2.0 y = 12.112x The RNA present in the 0.2ml sample The RNA present in the 0.4ml sample = 0.0128 mg = 0.0256 mg

The equation of the line for DNA is y = 1.014x The absorbance value is given by 1.014 x 0.0256 = 0.0260 Finding 10% of this value 0.1 x 0.0260 =0.00260 The RNA present is then corrected for by removing the confounding DNA variable 0.310 0.00260 = 0.307 Using the RNA equation of the line this corresponds to a value of 0.307/12.112 = 0.0254 mg of RNA in the 0.4mL sample The concentration was then 0.0251mg/0.4ml = 0.0635mg.mL-1 The solution was diluted 1 in 4 so the original solution RNA concentration was 0.0635 mg.mL-1 x 5 = 0.3175 mg.mL-1 The volume of the supernatant was 4.3ml so the total RNA content was 0.254mg.ml-1 x 4.3ml = 1.365mg of RNA This came from a 2ml sample of liver homogenate so Volume of the solution was 126ml 126ml x 0.6826mg.ml-1 = 86.01 mg The total RNA content of the Liver homogenate was 86.01mg The total mass of the liver was 12g so the concentration of RNA per gram of liver was = 7.17mg.g-1 = 0.6826mg.ml-1