Lecturer: Bahiya Osrah DNA ISOLATION

DEOXYRIBONUCLEIC ACID OR DNA The molecule that controls everything that happens in the cell. DNA contains the genetic code or commands that direct the activities of cells and ultimately, the body. DNA is present in all living things from bacteria to animals. In animals, it is found in almost all cell types, except red blood cells. DNA found within a nucleus in the cells. In order to extract DNA it’s necessary to denature and break down the cells to release DNA from these cells.

INTRODUCTION: The three main parts of the cell are : The nucleus, which holds DNA, The cell membrane, which surrounds and protects the cell, The cytoplasm, which is the jelly-like part of the cell between the membrane and the nucleus.

TO EXTRACT THE DNA WE NEED The basic aim: 1.To destroy cell membrane 2.To destroy the nucleus 3.To remove Protein 4.To collect DNA 5.To preserve DNA from breaking down

EXTRACTION OF DNA Extraction of DNA basically consists of Three major steps: 1. Preparation of a cell extract 2. Purification of DNA from cell extract 3. Measurement of purity and DNA concentration

FIRST: PREPARATION OF A CELL EXTRACT To extract DNA from tissue/cells of interest, the cells have to be separated and the cell membranes have to be disrupted. Reagents : Lysing step 1. EDTA (Ethylenediaminetetraacetate) which removes Mg+2 and Ca+2 ions These ions are essential for preserving the overall structure of the cell membrane To inhibit Dnase ( Mg+2 and Ca+2 are cofactors) 2. SDS (Sodiumdodecylsulfate), which aids in disrupting the cell membranes by removing the lipids of the cell membranes, and solubilized the proteins both are included in the extraction buffer which lysing the cells, 3. Protein enzyme or protease can be used to break down proteins.

The final step is the removal of insoluble cell debris by centrifugation leaving the cell extract as a reasonably clear supernatant. FIRST: PREPARATION OF A CELL EXTRACT

SECOND: PURIFICATION OF DNA FROM CELL EXTRACT cell extract now contain: DNA, protein and RNA. to remove these contaminants, leaving the DNA in a pure form we need to add: 1. Phenol: The standard way to de-proteinize (precipitate protein) a cell extract is to add phenol or a 111 mixture of phenol1 chloroform. These organic solvents precipitate proteins but leave the nucleic acids in aqueous solutions.The aqueous solution of nucleic acid can be removed with a pipette. 2. Ribonuclease enzyme: Ribonuclease enzyme to degrade RNA.

DNA Washing and elusion: DNA precipitation in ethanol or isopropanol (75%) DNA is insoluble in alcohol Alcohol used to wash and remove the previously added salts The result pellet will be washed by cold alcohol (cold to inhibit DNase DNA Re-suspention: TE buffer (Tris-EDTA) Usually in TE (Tris base –EDTA) buffer to preserve the DNA. EDTA is better than ddH2O. Store at 4ºC or -20ºC for longer time

OTHER METHODS FOR DNA EXTRACTION Kit “ all chemicals is mixed in reagents” Reagents: Proeinase K Genomic Lysis buffer Genomic Bind buffer DNA binding column 1xTE buffer Genomic wash buffer Ethanol 75% Elusion buffer Note: You can download the manual of DNA extraction from my website

THIRD: MEASUREMENT OF PURITY AND DNA CONCENTRATION DNA concentrations can be accurately measured by UV absorbance spectrometer at 260 nm. The amount of UV radiation absorbed by a solution of DNA is directly proportional to the amount of DNA sample UV absorbance can also be used to check the purity of a DNA preparation. With a pure sample of DNA the ratio of the absorbencies at 260 nm and 280nm (A 260/A 280)is 1.8 Ratios of less than 1.8 indicate that the preparation is contaminated, either with protein or with phenol.

DNA EXTRACTION FROM … FUN EXPERIMENT Can we extract DNA in our HOME!! YESSSSS

IS DNA IN MY FOOD??? DNA is present in the cells of all living organisms. The process of extracting DNA from a cell is the first step for many laboratory procedures in biotechnology. The scientist must be able to separate DNA from the unwanted substances of the cell gently enough so that the DNA does not denature (break up).

Each cell contains 9 feet of DNA In an average meal, you EAT approximately 55,000,000 cells This is equal to approximately 93,205 miles of DNA DNA FACTS

DNA DNA is found in the nucleus of all eukaryotic cells Most cells have the diploid 2n chromosome number Many plants are polyploid (contain several sets of chromosomes) Strawberrie s are octaploid 8n

DNA DNA is enclosed in a nuclear and a cell membrane made of phospholipids DNA is also coiled around proteins (hisones) Both the phospholipid layer and the proteins must be removed to see DNA

NUCLEUS with DNA

TO EXTRACT DNA, YOU MUST REMOVE … Cell membrane Cytoplasm Nuclear membrane Proteins

Shampoo or dishwasher soap: helps to dissolve the cell membrane, which is a lipid bilayer. Sodium chloride helps to remove proteins that are bound to the DNA. It also helps to keep the proteins dissolved in the aqueous layer so they don’t precipitate in the alcohol along with the DNA. Ethanol or isopropyl alcohol causes the DNA to precipitate. When DNA comes out of solution it tends to clump together, which makes it visible. The long strands of DNA will wrap around the stirrer or transfer pipet when it is swirled at the interface between the two layers. TO EXTRACT DNA, YOU NEED

HOW DO I BEGIN? Place the strawberry in a zip lock bag and remove the air Squash the berry with your hand to begin the process

CELL MEMBRANE Cell membranes are made of phospholipids Phospholipids won’t dissolve in water What do you use in your home to remove oils from your hands or your dishes? SOAP or DETERGENT!

SOAP & DETERGENT Soap gathers between oil and water capturing the oil in a bubble called a micelle When you wash your hands, the soap carries the oils away Dawn dishwashing works BEST to remove phospholipid membranes

WHY ADD DETERGENT? A cell's membranes have two layers of lipid (fat) molecules with proteins going through them.

WHY ADD DETERGENT? When detergent comes close to the cell, it captures the lipids and proteins.

SOAP & DETERGENT By adding a small amount of table salt (NaCl) to the soap solution, the solution can punch holes in the nuclear and cell membranes The soapy solution also helps removes proteins

ANOTHER WAY TO REMOVE PROTEINS histones DNA is tightly coiled around proteins called histones Proteins must be removed to see DNA Enzymes, like the ones found in meat tenderizer, can remove protein

EXTRACTING THE DNA The contents of the cell (organelles, proteins, etc.) must be separated from the DNA The larger cell parts can be removed by filtering the solid from the liquid

EXTRACTING DNA To see DNA, it must be extracted or “spooled” from the remaining liquid you filtered DNA dissolves in water, but NOT in alcohol Adding COLD ALCOHOL will cause DNA to precipitate (separate out) from the liquid filtrate

COLLECTING THE DNA The DNA will appear as a white precipitate once the alcohol is added HOLD THE TUBE by the TOP, not the bottom so the DNA strands won’t fragment from the heat of your hands!

SPOOLING THE DNA DNA is sticky and will adhere to other surfaces A glass stirring rod can be used to spool (remove) the DNA by using a turning motion

DNA ISOLATION USING STRAWBERRIES 1.Working in groups (for supervision), but each individual will do their own. 2.Place 1 strawberry in the plastic bag. Remove air and seal. 3.Mash strawberry for 2 minutes. 4.Add ~ 10 ml DNA Extraction buffer (Pre-measured). Remove air and seal. 5.Mash for 1 minute. 6.Pour solution through coffee filter into paper cup 7.Pour ~ 2 ml of this solution back into the 15 ml tube. It will be very red. 8.Pour ~ 4 ml (2X the volume of strawberry solution) of cold ethanol (found on freeze door in 50 ml tubes) so that it lays on top of the strawberry solution. (NOTE: can use 1X volume of room temperature isopropanol, rubbing alcohol, as a substitute) 9.If the tube just sits, there will be DNA at the interface with bubbles in it. If you gently rock the tube, more will come out of solution and eventually float to the top. (NOTE: if the DNA doesn’t come out of solution at home, add a little more salt)

Extraction buffer: 100 ml shampoo without conditioner or dishwashing liquid 900 ml distilled water 15 grams NaCl DNA ISOLATION USING STRAWBERRIES

LAB PROCEDURE: We have different tissues to isolate the DNA from We chose strawberries because: Ripe strawberries are an excellent source for extracting DNA because they are easy to pulverize and contain enzymes called pectinases and cellulases that help to break down cell walls. And most important, strawberries have eight copies of each chromosome (they are octoploid), so there is a lot of DNA to isolate.

MATERIAL AND METHOD DNA Extraction Buffer 100 ml (3/8 cup) shampoo (without conditioner) or 50 ml dishwasher detergent 15 grams sodium chloride (2 teaspoons) water to 1 liter

DIRECTIONS 1. Remove the green sepals from the strawberries. 2. Place strawberries into a Ziploc TM bag and seal shut. 3. Squish for a few minutes to completely squash the fruit. 4. Add 10 ml DNA Extraction Buffer (soapy salty water) and squish for a few more minutes. Try not to make a lot of soap bubbles. 5. Filter through a moistened paper towel set in a funnel, and collect the liquid in a clear tube. Do not squeeze the paper towel. Collect about 3 ml liquid. 6. Add 2 volumes ice cold isopropyl alcohol to the strawberry liquid in the tube. Pour the isopropyl alcohol carefully down the side of the tube so that it forms a separate layer on top of the strawberry liquid.

7. Watch for about a minute. What do you see? You should see a white fluffy cloud at the interface between the two liquids. That’s DNA! 8. Spin and stir the coffee stirrer or transfer pipet in the tangle of DNA, wrapping the DNA around the stirrer. 9. Pull out the stirrer and transfer the DNA to a piece of saran wrap or clean tube. The fibers are thousands and millions of DNA strands. 10. To view in a microscope, put the glob on a clean slide and gently tease/stretch apart using 2 toothpicks or dissecting pins. The fibers will be easier to see in the teased-apart area. DIRECTIONS