Molecular Biology 1 Lab Revision PDF

MOLECULAR Rev. BIOLOGY 1 BIO221 What Would We Learn Within the Practical Part? DNA & RNA DNA/RNA Isolation Assessment of DNA/RNA Quality & Quantity DNA/RNA Electrophoresis Othe...

MOLECULAR Rev. BIOLOGY 1 BIO221 What Would We Learn Within the Practical Part? DNA & RNA DNA/RNA Isolation Assessment of DNA/RNA Quality & Quantity DNA/RNA Electrophoresis Other Techniques  DNA Isolation Plant Cells Animal Cells  DNA Extraction from Plant Cells Introduction DNA extraction is the process of isolating DNA from cells to study it. Generally, it involves collecting the cells, breaking open them, removing proteins and other materials, and separating the pure DNA. X  Main Steps of DNA Isolation from Plant Cells 1 Sample Collection & Preparation Aim: Obtain a plant sample from which DNA will be extracted. Tomato Guava 2 Cell Lysis Aim: Break open the cells to release DNA into the solution by: Lysis Buffer Homogenization (for tougher tissues) Detergent (such as EDTA (Inhibits SDS or Triton X- DNases) 100) to disrupt the cell membrane. Salts like NaCl Mortar & Pestle *Both are used at the same time for tougher tissues Mechanism of Action of a Detergent incubate 3 Removal of Proteins and Other Contaminants Aim: Separate the proteins and other cellular debris from the DNA. After lysis, proteases are used (as Proteinase K) and RNases to digest proteins & RNAs that could interfere with the extraction. *Optional 4 Precipitation of DNA Aim: Isolate DNA from the aqueous phase. DNA will appear as a white, stringy material that can be spooled. Add cold ethanol or isopropanol to the solution, which causes DNA to precipitate out of the solution forming a pellet. 5 Washing the DNA Aim: Remove residual salts and contaminants by washing the DNA pellet with 70% ethanol to remove any remaining salts or organic compounds. Transfer some of the DNA fibers to an Epp tube. Centrifuge to make a DNA pellet Wash by adding 70% ethanol. Centrifuge and discard the ethanol, leaving a clean DNA pellet. 5 Resuspension and Storage Aim: Dissolve the purified DNA in a suitable buffer for storage and future use. Resuspend the DNA pellet in TE buffer (Tris- EDTA) or sterile distilled water. Store the DNA at 4°C for short-term use or -20°C or -80°C for long-term storage.  Summary of the DNA Isolation Process 1. Sample Collection: Gather cells or tissue. 2. Cell Lysis: Break open cells using lysis buffer and detergent. 3. Remove Contaminants: Separate proteins and debris 4. DNA Precipitation: Precipitate DNA with cold alcohol (ethanol/isopropanol). 5. Wash DNA: Use 70% ethanol to wash and purify DNA. 6. Resuspend DNA: Dissolve DNA in a buffer or water for storage.  DNA Extraction from Whole Blood BIO221  DNA Extraction from Whole Blood Whole blood means blood that is drawn directly from the body and contain all of its components like RBCs, WBCs & Platelets without any separation. XX X  Main Steps of DNA Isolation from Whole Blood 1 Sample Collection & Preparation Aim: Obtain a blood sample from which DNA will be extracted. Blood 2 RBCs Lysis Aim: Remove red blood cells using RBCs lysis buffer. RBCs Lysis Buffer Ammonium Chloride Potassium or Sodium EDTA (Hypotonic solution that Bicarbonate (Inhibits creates osmotic (Buffering agent that DNases) imbalance which maintains pH of the buffer disrupts RBCs only) & support lysis process)  Mechanism of Action of a RBCs Lysis Buffer H2O WBCs remain intact Centrifuge Add RBCs Lysis Buffer & incubate Water molecules Cell swells and enter RBC eventually burst X WBCs 3 WBCs Lysis Aim: Break open the cells to release DNA into the solution by: Lysis Buffer Detergent EDTA (such as SDS to (Inhibits disrupt cell membrane) DNases) Salt (NaCl) Mechanism of Action of a Detergent incubate 4 Removal of Proteins and Other Contaminants Aim: Separate the proteins and other cellular debris from the DNA. Transfer DNA to new tube After lysis, add ammonium acetate to precipitate proteins and then centrifuge to pellet proteins. Protein Decant DNA into a new tube. 5 Precipitation of DNA Aim: Precipitation of DNA from the supernatant by rubbing ethanol or isopropanol. Add cold ethanol or isopropanol to the supernatant to precipitate DNA and then centrifuge to X DNA pellet DNA. 6 Washing the DNA Aim: Remove residual salts and contaminants by washing the DNA pellet with 70% ethanol to remove any remaining salts or organic compounds. Wash the DNA by adding 70% ethanol and pipetting up and down. Then, Centrifuge and discard the ethanol supernatant, leaving a clean DNA pellet. 7 Resuspension and Storage Aim: Dissolve the purified DNA in a suitable buffer for storage and future use. Resuspend the DNA pellet in TE buffer (Tris- EDTA) or sterile distilled water. Store the DNA at 4°C for short-term use or -20°C or -80°C for long-term storage. Summary of the Whole Blood DNA Isolation Method 1. Sample Collection: Collect whole blood sample. 2. RBCs Lysis: Remove red blood cells. 3. WBCs Lysis: Break open cells using lysis buffer. 4. Remove Protein Contaminants: Separate proteins and debris. 5. DNA Precipitation: Precipitate DNA with ice-cold alcohol (ethanol/isopropanol). 6. Wash DNA: Use 70% ethanol to wash and purify DNA. 7. Resuspend DNA: Dissolve DNA in a buffer or water for storage.  DNA Extraction from Cultured Animal Cells & Animal Tissues BIO221 DNA Extraction from Cultured Animal Cells & Animal Tissues by Phenol:Chloroform:Isoamyl Alcohol (PCI) Method DNA is extracted by many methods, but PCI method is the classic and most widely used for DNA Isolation due to its high DNA yield and quality. After lysis of cells and releasing their components, PCI is used as follow: Phenol (25) Chloroform (24) Isoamyl Alcohol (1) Phenol is hydrophobic Improves the Reduces and denser than water. efficiency and clarity foaming during So, it interacts with and of phase separation the mixing of precipitate hydrophobic by making proteins phenol and parts of proteins after and lipids more chloroform denaturing them and soluble in the also dissolves lipids. organic phase  PCIA DNA Extraction Method Main Steps Cultured cells Collect Cells and Pellet by Centrifugation Add PCI to Decant the lysate supernatant (Aquas Phase) (Interface) (Organic Phase) Add lysis buffer 3 phases formed Take the aqueous Wash by 70% Add ice-cold ethanol and incubate phase to a new tube ethanol & Store or isopropanol to precipitate DNA in TE or Water  PCIA DNA Extraction Method Main Steps Animal Tissue Put the tissue in a mortar, add lysis buffer and grind Take part of the homogenate into a microfuge tube Add PCI to Decant Incubate the lysate supernatant (Aquas Phase) (Interface) (Organic Phase) 3 layers formed Take the aqueous Add ice-cold ethanol Wash by 70% phase to a new tube or isopropanol to ethanol & Store precipitate DNA in TE or Water  Summary of the DNA Extraction Process 1. Sample Collection & Preparation: Gather cells in case of using cultured cells and homogenize tissue in case of using a tissue. 2. Cell Lysis: Break open cells using lysis buffer. 3. Remove Contaminants: Separate proteins and debris (e.g., using phenol-chloroform-isoamyl alcohol). 4. DNA Precipitation: Precipitate DNA with ice-cold alcohol (ethanol/isopropanol). 5. Wash DNA: Use 70% ethanol to wash and purify DNA. 6. Resuspend DNA: Dissolve DNA in a buffer or water for storage. Thanks!