cell biology lab report on LAB 1A: DNA Isolation from Wheat Germ LAB 1B: Spectrophotometric Quantification of DNA and Protein

Preparation for Assays Prior to the lab, a subsample of your isolated DNA was prepared by dissolving 30 mg in 30 ml of 0.01N NaOH Serial Dilutions ● Performed by doing a step-wise transfer of solution, resulting in the production of various concentrations ● Used to produce a standard curve for determining the concentration of an unknown sample Protein Standard x2 ● 2-fold dilution series of BSA BSA standard solution = 3 mg/ml Protein Standard x2 ● 2-fold dilution series of BSA Concentration (mg/ml) 3.0 1.5 .75 .375 .188 .094 01/2 1/2 1/2 1/2 1/2 All tubes have final volume of 3 ml DNA Standard ● 2-fold dilution series of DNA DNA standard solution = 1 mg/ml DNA Standard ● 2-fold dilution series of DNA Concentration (mg/ml) 1.0 .5 .25 .125 .0631/2 1/2 1/2 1/2 0 All tubes have final volume of 2 ml Internal Controls ● Sample with a known concentration ● Used to ensure that your assay is functioning properly ● Protein: 0.375 mg/ml BSA ● DNA: 0.25 mg/ml for Dische & 0.025 mg/ml for UV ● Prepare with same final volume as for the relevant standards Unknown Samples ● Using the DNA isolated from wheat germ in the previous lab, and solubilized in advance ● Use a number of dilutions in order to ensure one falls within range of your standards (prepare in same final volume) ● For protein: undiluted, 2-fold, & 5-fold ● For DNA: undiluted for Dische, and 5-fold & 10-fold for UV Part B: Colorimetric Assays ● Utilize spectrophotometry in the visible light range ● Based on production of a coloured product due to a chemical reaction with the compound of interest ● Pro: highly specific ● Con: destruction of sample, use of caustic reagents (in our assays) Colorimetric Assays for Protein ● Both based on interaction of copper ions with peptide bonds to produce a coloured product ● Specific need for peptide bonds means that even amino acids are unreactive Biuret Assay ● Results in the production of a purple compound ● Add 5 ml Biuret reagent to all tubes & incubate 10 min at room temp Read at 540 nm Lowry Assay ● Add 5 ml Lowry reagent to all tubes & incubate 10 min at room temp ● Add 1 ml Folin reagent & incubate 15 min at room temp: results in green product Read at 500 nm Biuret vs Lowry ● Additional reagent in Lowry assay results in a more sensitive assay – Can detect lower amounts of protein ● Lowry assay is more sensitive to certain assay conditions – Can result in reduced accuracy Dische Assay for DNA ● Based on the ability of diphenylamine to react with deoxyribose sugars ● DNA is partially hydrolyzed with heat, which can then interact with the reagent, producing a blue product Read at 600 nm Part C: UV Assays ● Utilize spectrophotometry with UV light ● Aromatic compounds will absorb light, so increased amounts of these compounds results in an increased absorbance reading ● Pro: no reagents, so sample can be reused ● Cons: turbid sample results in light- scattering & nonspecific detection UV Assay for Protein ● Based on detection of the three aromatic amino acids: phenylalanine, tyrosine, and tryptophan ● Maximum absorbance at 280 nm ● Detection based on relative quantity of these specific amino acids, not total amount of protein ● BSA standard usually used: relatively average amounts of these amino acids UV Assay for DNA ● Based on detection of the nitrogenous bases of DNA ● Maximum absorbance at 260 nm ● Additional concern is the hyperchromatic effect: partially unwound DNA will result in increased absorbance Sample Purity ● All of DNA, RNA, & protein can be detected at both 260 and 280 nm ● Determine sample purity by determining the 260/280 ratio – Pure DNA has a ratio of 2.05 – Acceptably pure DNA has a ratio of 1.85 or higher Data Analysis ● Standard curve preparation ● Determination of unknown concentrations ● Determining actual concentration:

which results to use?

● Determining quantity of actual DNA isolated Standard Curves ● Independent variable on X-axis, dependent variable on Y-axis Don’t forget to include units for concentration, and the wavelength for absorbance! Standard Curves ● Always exclude values outside of the linear range Standard Curves ● Include a best-fit straight line forced through zero y = mx + b means y = (slope)(x) + y-intercept Since b = 0, Absorbance = (slope)(conc) Determining Unknown Concentrations ● Use the y=mx equation generated by your standard curve ● The absorbance of your sample is simply divided by the slope ● You need to correct each sample for dilution!

● Average all samples with an absorbance that falls within the linear range of the standard curve – Exception: if there seems to be a human error in a single dilution, you may also exclude it What is the Actual Concentration?

● Look at the reliability of each assay, to determine whether it should be included in your final determination – Was the internal control concentration accurate?

– Were the corrected concentrations for your different dilutions similar or was there significant variation?

– For the UV assays: what was the 260/280 ratio? Was the sample pure enough for it to be reliable? Quantity of Isolated DNA First, determine the ratio of actual DNA :

= determined concentration subsample concentration = (mg/ml) 30 mg / 30 ml This is 1 mg/ml for your report, but this might not always be the case! Final determined DNA concentration from assays Quantity of Isolated DNA Then, determine the actual amount of DNA isolated :

= dry weight of DNA isolated (mg) x actual DNA ratio Provided in the data file on Nexus in grams Finally, determine the amount of DNA isolated / g of wheat germ We started with 5g of wheat germ Lab Report ● Lab 1 is to be submitted as a formal lab report (see the General Lab Report Guidelines AND the Lab 1 Report Requirements ) ● Your lab report must be submitted through Nexus by 11:59 PM on October 8 th as a pdf file ● It is worth 7.5% of your final grade! Results ● Data for your report will be available on Nexus midday on Thursday ● Ensure that you follow the guidelines for proper table and figure preparation – Your reader should understand what they contain without having to refer to the text – Appropriate titles (above tables and below figures), followed by captions containing sufficient detail – Inclusion of appropriate labels and units ● All results included in tables and figures must be included in the associated text and refer to the table and figure number. The tables/figures support your text! Results ● Standard curves for each colorimetric assay (you do not need tables of standard absorbances) ● Tables including the absorbance, calculated concentration, and corrected concentration for both unknowns and internal controls. Include the determined concentration in your captions along with a statement as to any excluded dilutions.

● The 260/280 ratio should be included in your text ● Include your determination of the total amount of DNA isolated (mg DNA / g wheat germ). You should describe how you calculated this value in the text, along with numbers used.

● You do not need to include any sample calculations. Discussion ● Maximum 750 words.

● Start by discussing your results, including your findings as to reliability of the assays used. Refer to published works as to the limitations of each assay.

● Discuss the potential errors that may have led to any discrepancies, as well as means by which your assays or techniques could be improved ● Are there alternative techniques for isolation and/or quantification?

● Finish with a concluding paragraph. Summarize your overall findings and conclusions. Which assays were most reliable? End with a concluding statement which provides importance to your work. Abstract ● Maximum 250 words ● This is a summary of your entire report, and is best written last ● Include a statement of purpose/importance, brief description of the methods employed, key specific results (including values), and your overall conclusions. References ● The guidelines on Nexus provide an example of appropriate referencing ● While I prefer the numbered superscript citation methods, the author/year format is also acceptable ● Complete references must be included within a reference section, not as footnotes ● Remember: all information from another source must be referenced!