8 FAQs For DNA Extraction

The Extraction Of Plasmid Dna Is An Essential Experimental Skill In DNA Technology. Isolation Of Plasmid DNA Is Generally Divided Into Three Steps: 1. Cultivate Bacteria To Amplify Plasmids; 2. Collect And Split Bacteria; 3. Isolation And Purification Of Plasmid DNA Alkali Splitting Method Is A Common Extraction Method. During The Process Of Plasmid Extraction, Due To Mechanical Force, PH, Reagents, Etc., The Plasmid DNA Chain May Break. Therefore, Most Of The Crude Extracts Of Plasmids Contain Three Configurations Of Plasmids: Covalently Closed Circular DNA (cccDNA): Two Strands Of The Plasmid Are Not Broken; There Are Always Some Problems In The DNA Extraction Of Super Helix Open Loop DNA (ocDNA), Which Leads To No Way To Start The Experiment. Let's Analyze Some Common Problems: Why Precipitate DNA With Anhydrous Ethanol? The Advantage Of Ethanol Is That It Can Be Miscible With Water At Any Ratio. Ethanol And Nucleic Acid Will Not Have Any Chemical Reaction. When Ethanol Is Added, It Will Seize The Water Molecules Around DNA, Making DNA Dehydrated And Easy To Polymerize. In General, 95% Ethanol Can Be Used To Replace Absolute Ethanol When DNA Is First Precipitated, And Absolute Ethanol Should Be Used For The Final Precipitation Step. DNA Can Also Be Selectively Precipitated With 0.6 Times The Volume Of Isopropanol. Generally, It Can Be Left At Room Temperature For 15-30 Minutes. When DNA Is Precipitated With Absolute Ethanol, Why Must NaAC Or NaCL Be Added Until The Final Concentration Reaches 0.1-0.25MOL/L? In The DNA Solution With A Ph Of About 8, The DNA Molecules Are Negatively Charged. Add A Certain Concentration Of NaAC Or NaCL To Neutralize The Negative Charge On The DNA Molecules, Reduce The Repulsive Force Of The Same Charge Between DNA Molecules, And Easily Polymerize With Each Other To Form DNA Sodium Salt Precipitation. When The Concentration Of The Added Salt Solution Is Too Low, The DNA Precipitation Is Incomplete, Otherwise, It Is Difficult To Collect DNA. In The Process Of DNA Precipitation, Due To The Existence Of Excessive Salt Impurities, Which Affect DNA Digestion And Other Reactions, It Is Necessary To Wash Or Re Precipitate. Why Is TE Buffer Generally Used In The Process Of DNA Preservation Or Extraction? In The Gene Operation Experiment, The Main Principle Of Selecting Buffer Solution Is To Consider The Stability Of DNA And The Buffer Solution Components Do Not Produce Interference. Therefore, Tris HCL (pKa=8.0) Buffer System Is Used. Since The Buffer Solution Is TrisH/Tris, And There Is No Interference Of Metal Ions, EDTA In TE Buffer Solution Can More Stabilize The Activity Of DNA When Extracting Or Storing DNA. After Ribonuclease Is Added To Degrade Ribonucleic Acid, SDS And Kac Should Be Used To Treat It Once To Make The Precipitation Better And Complete. How To Choose The Concentration Of Polyethylene Glycol (6000) To Precipitate DNA? When PEG (6000) Is Used To Precipitate DNA, The Concentration Of PEG Used For DNA Molecules Of Different Sizes Is Different. The Concentration Of PEG Is Low, And The Molecular Weight Of DNA Is Large. The Concentration Of PEG Required For Macromolecules Is Only About 1%, While The Concentration Of PEG Required For Small Molecules Is About 20%. Why Do You Add A Small Amount Of Isosorbitol When Extracting DNA With Phenol And Chloroform? When Extracting DNA, In Order To Mix Evenly, The Radiant Must Be Violently Shaken For Several Times. The Addition Of Isosorbitol Can Reduce The Surface Tension Of Molecules And Reduce The Generation Of Bubbles During Oscillation; Generally, The Ratio Of Chloroform And Isopentanol Is 24:1, And Phenol Can Also Be Used. When DNA Is Extracted To Remove Protein, Since Phenol And Chloroform Are Mutually Soluble, Chloroform Can Be Used To Denature The Protein For The Second Time, And Then The Phenol Can Be Taken Away Together. It Is Also Possible To Mix Phenol With Chloroform (1:1) During The Second Extraction. Why Should We Saturate Phenol With Tris Aqueous Solution Of PH8? Because Phenol And Water Are Mutually Soluble. The Purpose Of Phenol Water Saturation Is To Prevent It From Absorbing The Water Containing DNA In The Sample And Reduce The Loss Of DNA During DNA Extraction. Adjust The PH To 8 With Tris Because DNA Is Relatively Stable Under This Condition. Can I Use The Pink Phenol? Phenols Stored In The Refrigerator Are Easy To React With Oxygen In The Air And Turn Pink. Such Phenols Are Easy To Degrade DNA. How Can We Keep Phenols From Being Oxidized By Air? Add Mercaptoethanol And 8-hydroxyquinoline Until The Final Concentration Is 0.1%. 8-hydroxyquinoline Is A Light Yellow Solid Powder, Which Can Not Only Resist Oxidation, But Also Inhibit The Activity Of Dnase To Some Extent. Phenol Saturated With Tris PH8.0 Aqueous Solution Is Best Sub Packed In Small Brown Reagent Bottles, Covered With A Layer Of Tris Aqueous Solution Or TE Buffer To Isolate Air, Or Filled With Nitrogen To Prevent Oxidation Due To Contact With Air. Then Store It In A Refrigerator At 4 ℃ Or - 20 ℃, Which Can Be Kept For Several Months Without Discoloration.