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

In order to ensure the proper functioning of your assay, you will include an internal control .

This is a sample of known concentration, which allows you to assess whether you are obtaining reliable results from your assay.

Proteins are high molecular weight polymers of amino acids . The amino acids are linked by peptide bonds to form a linear chain of amino acids, or a polypeptide, which takes on a three-dimensional shape in solution. As these peptide bonds are unique to proteins, colorimetric assays that utilize a chemical reaction to detect them are extremely specific. Several assays are based on the ability of copper ions to form a purple-colored complex with two or more peptide bonds when in alkaline conditions. The amount of these complexes formed can then be determined by visible light spectrophotometry (usually at 500- 700 nm) and, using a standard curve constructed with a protein of known concentration, the concentration of the unknown protein sample can be estimated. In this lab, we will utilize two colorimetric assays to determine protein concentration: the Biuret and Lowry methods. Both methods are based on the principles outlined above, but since the Lowry method uses an additional reagent to enhance color development, it is a more sensitive assay (detects lower concentrations of protein). However, the reagents used in the Lowry assay also result in it being more susceptible to reduced accuracy in certain assay conditions.

Each nucleotide of DNA is comprised of a phosphate group, deoxyribose sugar, and nitrogenous base. These nucleotides are polymerized into a long, linear molecule through phosphodiester bonds between the phosphate groups and sugars. The colorimetric assay that we will utilize in this lab is the Dische diphenylamine reaction , which allows the detection of deoxyribose sugars . When DNA is partially hydrolyzed through boiling, the diphenylamine reagent reacts with the sugar and turns blue, allowing its quantification with a visible light spectrophotometer. While the Dische reaction can detect any 2-deoxypentose sugar, there is very little interfering material found within the cell, so it is a relatively specific test for DNA concentration.

Finally, we will utilize UV spectrophotometry to determine both the protein and DNA content in our samples. In both cases, the assay utilizes the ability of aromatic compounds to absorb light at a given wavelength. In the case of proteins, three different aromatic amino acids can be found ( tryptophan, tyrosine, and phenylalanine ), each with their own unique absorbance spectra (see Figure 1 on the following page ). As the optimal absorbance for proteins occurs at 280 nm , this wavelength is used for protein concentration assays. On the other hand, DNA contains nitrogenous bases , which absorb strongly at 260 nm . However, both molecules, as well as RNA and free monomers, absorb light at either wavelength, making contamination a serious concern for accurate measurement. We will be using the NanoDrop TM to perform these assays, which allows for 2 μ L samples to be assessed. Figure 1: The ultraviolet absorption spectra of tryptophan, tyrosine, and phenylalanine, in aqueous solutions.

UV assays also may result in inaccurate measurements if the sample is too concentrated or turbid (due to light-scattering effects ). In the case of measuring DNA concentration, the hyperchromatic effect must also be considered, which results in an increase in absorbance when the DNA becomes unwound due to even partial hydrolysis. In order to determine the validity of DNA concentration results, UV testing is associated with the 260/280 ratio . A pure sample of DNA will have a ratio of 2.05, with any ratio greater than 1.85 deemed “acceptably pure”.

In this lab, we will determine protein concentration using the Biuret, Lowry, and UV methods. We will utilize both the Dische and UV methods of DNA concentration determination. For the three colorimetric assays, standard curves will need to be prepared using either known protein (bovine serum albumin) or DNA concentrations (see Appendix B ).

These standard curves will be used to determine the concentration of DNA and protein in your wheat germ DNA sample. We will also use internal controls to ensure the validity of your results. The NanoDrop TM will be used for UV-based determination of DNA and protein concentrations, as well as the 260/280 ratio to analyze the purity of the DNA isolated from wheat germ in Lab 1A . 2 PROCEDURES A. Preparation of standards, controls, and wheat germ DNA 1. Protein standards a. Label 2 sets of 7 test tubes (14 in total), 1 through 6 and BLANK, with a grease pencil or felt marker. One set will be used for each of the colorimetric protein assays: Biuret and Lowry.

b. Pipet 6.0 mls of the bovine serum albumin stock solution provided (3.0 mg/ml) into each tube 1 . Pipet 3 mls of distilled water into the remaining 12 tubes .

c. Prepare your first set of standards as shown below. Pipet 3 mls of the albumin stock from tube 1 into tube 2 . Mix the contents by pipeting up and down. Then pipet 3 mls of the contents of tube 2 into tube 3 . Mix. Repeat this process until you have mixed tube 6. Pipet 3 mls from tube 6 and discard. DO NOT transfer any liquid into the BLANK tube, as this is should contain 0 mg/ml of albumin.

d. Repeat step c for the other set of tubes . The tubes now contain 3 mls each of albumin concentrations (mg/ml) as follows:

Tube 1 3.0 Tube 2 1.5 Tube 3 0.75 Tube 4 0.375 Tube 5 0.188 Tube 6 0.094 BLANK 0 2. DNA Standard: The DNA standards are prepared using a serial dilution in screw-cap tubes, similar to how this was performed for the protein standards, as depicted below:

3 The tubes now contain 2 mLs each of DNA concentrations (mg/ml) as follows:

Tube 1 1.0 Tube 2 0.5 Tube 3 0.25 Tube 4 0.125 Tube 5 0.0625 BLANK 0 3. Controls and wheat germ DNA You will be taking both DNA and protein measurements from your wheat germ DNA sample prepared in Lab 1A . An information sheet is provided with your sample to explain how it was prepared (you need this info for your assignment). Your wheat germ DNA sample should be filtered before use with the Whatman 3MM filter paper provided. You will also need to include the internal controls for protein or DNA in each assay. Be sure to clearly label each of your tubes to avoid confusion later on. a. Prepare your wheat germ sample and internal control for your protein assays. Label two sets of regular test tubes and one set of small test tubes for each of the preparations listed below.

i. Undiluted: 3 mL of wheat germ sample in each of two regular and one small test tubes.

ii. 2-fold dilution : Combine 5 mL of wheat germ sample and 5 mL of water in a regular test tube. Mix by pipeting up and down and transfer 3 mL each to one regular and one small test tube. Discard 1 mL from the original tube.

(see diagram on the right) iii. 5-fold dilution : Prepare as for the 2-fold dilution, but combine 2 mL of wheat germ sample and 8 mL of water. Mix and transfer 3 mL to each of the 2 other tubes. Discard 1 mL from the original tube.

iv. Control protein: Add 3 mL of your 0.375 mg/ mL BSA protein internal control to each of two regular and one small test tube.

You should have a total of 12 tubes (3 each of undiluted, 2-fold, 5-fold, and control), each containing 3 mL.

b. Prepare your wheat germ sample and internal control for your DNA assays :

i. Undiluted for the Dische assay : 2 mL of wheat germ sample into a large test tube with screw cap ii. 5-fold dilution for the UV assay : Use a small test tube. Combine 1 mL of wheat germ sample with 4 mL of water. Mix by pipeting up and down. iii. 10-fold dilution for the UV assay : Transfer 1 mL of your 5-fold dilution to a small test tube containing 1 mL of water. Mix by pipeting up and down. 4 iv. Control DNA for Dische assay : Add 2 mL of the 0.25 mg/ml DNA internal control into a large test tube with screw cap v. Control DNA for the UV assay : Add 2 mL of the 0.025 mg/ml DNA internal control into a small test tube.

You should have a total of 5 tubes. 2 for the Dische assay in large tubes with screw caps and 3 for the UV assay in small test tubes.

B. Performing the colorimetric assays Each of the three assays will be performed using the visible light spectrophotometers on your benchtop.

1. The Biuret test for protein a. Collect one series of protein standards (tubes 1-6 and BLANK), one set of your wheat germ sample dilutions (undiluted, 2-fold, and 5-fold), and one protein control. In total, you should have 11 regular sized test tubes .

b. Add 5 mls of Biuret reagent to each tube, including the BLANK . Vortex briefly.

c. Let these samples stand at room temperature for 10 minutes .

d. Set the spectrophotometer to read absorbance (A) at a wavelength of 540 nm .

e. After the incubation period, pour the contents of the BLANK into a cuvette and zero the instrument. You will then use a new cuvette and, starting with the most dilute of the Biuret standards, obtain absorbance readings for all of the standards. Next, measure your wheat germ dilutions (again starting with the most dilute and a new cuvette) and finally your control (in a fresh cuvette). If performing the experiment yourself, you would record your readings in the table below. Data for all of the assays will be posted on Nexus for your report.

BSA Standard (mg/ml) Absorbance (@540 nm) Sample Absorbance (@540 nm) 0 .094 5-fold dilution of wheat germ 0 .188 2-fold dilution of wheat germ 0 .375 Undiluted wheat germ 0 .750 1 .500 Internal control (0.375 mg/ml) 3 .000 5 2. The Lowry test for protein CAUTION: Folin reagent is toxic and should be handled with care.

a. Collect one series of protein standards (tubes 1-6 and BLANK), one set of your wheat germ sample dilutions (undiluted, 2-fold, and 5-fold), and one protein control . In total, you should have 11 regular sized test tubes .

b. Add 5 mls of Lowry reagent to each tube, including the BLANK . Vortex briefly.

c. Incubate at room temperature for 10 minutes.

d. Add 1.0 ml of Folin reagent to each tube, including the BLANK . Vortex. Let the tubes stand at room temperature for 15 minutes.

e. Set the spectrophotometer to read the absorbance (A) at a wavelength of 500 nm .

f. After the incubation period, pour the contents of the BLANK into a cuvette and zero the machine. You will then use a new cuvette and, starting with the most dilute of the Lowry standards, obtain absorbance readings for all of the standards. Next, measure your wheat germ dilutions (again starting with the most dilute and a new cuvette) and finally your control (in a fresh cuvette). 3. The Dische diphenylamine reaction.

BE CAREFUL! Dische reagent contains concentrated sulfuric acid and is caustic. Wear PPE! Keep all samples under the snorkels at all times. Ensure that the water on the hot plate is boiling prior to adding the reagent.

a. Collect the large, screw-cap test tubes. This should include your DNA standards (1-5 and BLANK), your undiluted wheat germ sample, and your control DNA . You should have a total of 8 tubes .

b. Add EXACTLY 4 mls of the Dische diphenylamine solution to each tube.

c. With the caps on , mix well by vortexing carefully. Loosen the caps of the test tubes, and place them in the boiling water for 10 minutes.

d. After 10 minutes, place the tubes into an ice bath to cool them. The tubes will cool rather quickly in the ice so do not leave them in too long.

e. Set the spectrophotometer to read absorbance (A) at a wavelength of 600 nm . Once the tubes have cooled to approximately room temperature, pour the contents of the BLANK into a cuvette and zero the machine. You will then use a new cuvette and, starting with the most dilute of the Dische standards, obtain absorbance readings for all of the standards. Next, 6 measure your wheat germ sample and finally your control. Use fresh cuvettes for your sample and your control. C. Ultraviolet spectrophotometry Once everyone in your group has completed the colorimetric assays for Part B, collect your UV assay (small) test tubes which were prepared during Part A. This should include your wheat germ sample dilutions (3 for protein and 2 for DNA), protein control , and DNA control . You should have a total of 7 tubes. Standards are programmed into the NanoDrop TM and therefore do not need to be included. The machine will automatically calculate the concentrations of DNA and protein present in your diluted samples, as well as the 260/280 ratio for DNA purity.

Prior to the Lab 2B Tutorial , please view the Spectrophotometry video and review Appendix B: Spectrophotometry and Standard Curves.

Lab 1 Report This lab will require the submission of a formal lab report worth 7.5% of your final grade in the course, and is due by October 8 th at 11:59 PM . The General Lab Report Guidelines can be found in the Lab Manual & Info folder. The specific Lab 1 Report Requirements are available in the Lab 1 folder. Standard curve preparation and calculations will be discussed during the Zoom tutorial.

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