BIOL 3120 Cell Biology Lab - Lab 6 PDF

BIOL 3120 - Cell Biology Lab Lab 6. Genomic DNA extraction; & Total RNA Extraction (Demo) Rubaia Tasmin 10/14/2024 Email: [email protected] 1 Today’s Lab Objectives 1. Understand the principles of genomic DNA extraction from pla...

BIOL 3120 - Cell Biology Lab Lab 6. Genomic DNA extraction; & Total RNA Extraction (Demo) Rubaia Tasmin 10/14/2024 Email: [email protected] 1 Today’s Lab Objectives 1. Understand the principles of genomic DNA extraction from plant tissue 2. Extract genomic DNA from plant tissue (Arabidopsis thaliana leaf sample) 3. Measure the concentration and assess the purity of the genomic DNA extracts 2 Principal of DNA extraction in plants Add isopropanol to precipitate and 70% ethanol Disrupt the cell wall Separate DNA to wash the DNA and plasma from organic phase Add water to solubilize the membrane DNA. The polar water molecules surrounds the charged regions of DNA, allowing it to dissolve. 3 Extraction Buffer 1.50 mM Tris-HCl: maintain the pH at the optimal condition for DNA extraction 2.10 mM EDTA : EDTA binds to metal ions, like magnesium and calcium, which are cofactors of DNase; thus preventing the degradation of DNA. 3.100mM NaCl: dissolves the DNA and maintains its stability 4.1.0 % SDS: SDS causes protein denaturation and disrupts the interactions between proteins and lipids in cell membranes, causing the cell to break down and release its contents. 5.10mM β-mercaptoethanol: a strong reducing agent that breaks the disulfide bonds of proteins, making them insoluble in water to obtain pure DNA that is free from protein contamination. 4 Potassium acetate and Isopropanol /ethanol Potassium acetate (Bio 101 Solution III) Creates a high-salt environment, and neutralizes the high pH of the lysis solution, facilitating the precipitation of SDS-bound proteins and other cellular debris. Isopropanol / ethanol The isopropanol facilitates the interaction of K+ or Na+ with the PO3− groups on the sugar phosphate backbone of nucleic acids. This interaction makes the DNA molecule less soluble in water, precipitating the DNA. 5 DNA qualification and quantification 1. The concentration and purity can be measured by NanoDrop spectrophotometer. 2. Nucleic acids and proteins have an absorbance maxima at 260nm and 280nm, respectively. Pure DNA will have an A260/A280 ratio of 1.8–2.0. 3. Lower 260/280 ratios usually indicate that a sample is contaminated by residual phenol, sugar, or the reagents used in the 6  Lab 6 Report Requirements 1. Introduction: Principal of Genomic DNA extraction and purpose of the lab. 2. Materials: All the chemicals, along with concentration, and instruments used. 3. Methods: Brief steps for the experiment you did. 4. Results: The concentration of your extracted genomic DNA The 260/280 ratio of the genomic DNA Keep the DNA in the freezer for next lab 5. Discussion/conclusion: Interpret your results. Discuss unexpected findings. What improvements can you make? What is your conclusion? What is the significance of the lab? 6. FileLab manual name for yourquestions & underlined questions lab report submission- BIOL3120_Section_Lastname_Firstname_ None Lab 6​ Send me the Word document by email 7  BIOL 3120 - Cell Biology Lab Total RNA Extraction (Demo) Zijie Zhang 10/08/2024 Email: [email protected] 8 Today’s Lab Objectives (cont.) 1. To learn the precautions for working with RNA. 2. To learn the principle of RNA extraction 9 Precautions for working with RNA  Keep the RNA samples intact by placing on ice while in use - For long term storage: store RNA samples in –80°C  Control the source of RNase - Use RNase-free pipette tips and microcentrifuge tubes  Clean work area and pipettes with RNaseAway  Wear clean gloves, masks and lab coat  Inactivate the RNase using DEPC water 10 Principle of RNA extraction in plants 11 RNA assessment https://www.promega.com/resources/pubhub/methods-of-rna-quality- assessment/ 12