Date conversion 20.02.2019 Size 9,94 Kb.
Outline Purpose of DNA extraction Review the main steps in the DNA extraction protocol and the chemistry involved in each step Purpose of DNA Extraction Basic Protocol Most DNA extraction protocols consist of two parts A technique to lyse the cells gently and solubilize the DNA Enzymatic or chemical methods to remove contaminating proteins, RNA, or macromolecules In plants, the nucleus is protected within a nuclear membrane which is surrounded by a cell membrane and a cell wall. Four steps are used to remove and purify the DNA from the rest of the cell. Lysis Precipitation Wash Resuspension A comparison of DNA extraction methods used in research labs as opposed to classroom labs Research : grind in Liquid N2 and use detergent Lysis : phenol/chloroform extraction Precipitation Part I to get rid of proteins : addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA Precipitation Part II : addition of ethanol Precipitation Part III to pull DNA out of solution DNA Wash and resuspend: is washed in ethanol, dried, and resuspended in H20 or TE buffer. Classroom : grind in mortar/pestel and use detergent Lysis : NONE (chemical are too dangerous!) Precipitation Part I : addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA Precipitation Part II : addition of ethanol to pull DNA out of solution Precipitation Part III DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer. Wash and resuspend: LYSIS: In DNA extraction from plants, this step commonly refers to the breaking of the cell wall and cellular membranes (most importantly, the plasma and nuclear membranes) The cell wall (made of cellulose) is disrupted by mechanical force (for example, grinding the leaves) Then the addition of a detergent in the which breaks down the cell membranes Detergents are able to disrupt membranes due to the amphipathic (having both hydrophilic and hydrophobic regions) nature of both cellular membranes and detergent molecules. The detergent molecules are able to pull apart the membranes The end result of LYSIS is that the contents of the plant cells are distributed in solution. PRECIPITATION (In a research lab): This a series of steps where DNA is separated from the rest of the cellular components In a research lab, the first part of precipitation uses phenol/chloroform to remove the proteins from the DNA Phenol denatures proteins and dissolves denatured proteins. Chloroform is also a protein denaturant THIS STEP CANNOT BE PERFORMED IN CLASSROOM LABS!! The second part of research lab DNA precipitation is the addition of salts The salts interrupt the hydrogen bonds between the water and DNA molecules. The DNA is then precipitated from the protein in a subsequent step with isopropanol or ethanol In the presence of cations, ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution. The DNA is pelleted by spinning with a centrifuge and the supernatant removed PRECIPITATION (In a classroom lab): This a series of steps where DNA is separated from the rest of the cellular components In a classroom lab, DNA precipitation involves the addition of salts The salts interrupt the hydrogen bonds between the water and DNA molecules. The DNA is then precipitated from the protein in a subsequent step with isopropanol or ethanol In the presence of cations, ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution. The DNA is pelleted by spinning with a centrifuge and the supernatant removed Note: because this protocol does not use phenol/chloroform, the DNA extracted in a classroom lab is not as “clean” as the DNA extracted in a research lab! Washing: Washing: The precipitated DNA is laden with acetate salts. It is “washed” with a 70% ethanol solution to remove salts and other water soluble impurities but not resuspend the DNA. Resuspension: The clean DNA is now resuspended in a buffer to ensure stability and long term storage. The most commonly used buffer for resuspension is called 1xTE Washing and Resuspension: Centrifuge to separate the solids from the dissolved DNA Precipitate the DNA using isopropanol Centrifuge to separate the DNA from the dissolved salts and sugars Overview of DNA Extraction Checking the Quality of your DNA The product of your DNA extraction will be used in subsequent experiments Poor quality DNA will not perform well in PCR You will want to assess the quality of your DNA extraction using the following simple protocol: Mix 10 µL of DNA with 10 µL of loading buffer Load this mixture into a 1% agarose gel Analyze results (the following slides provide guidance) Below is an agarose gel that has 5 genomic DNA samples from various plants. Note that the DNA runs at a very high molecular weight and as a clear, thick band. This DNA was extracted in a research lab under optimal conditions If properly done, genomic extraction should result in bright bands in the very high base pair range of a gel electrophoresis. Sizes of Genomic DNA for various Species in kbp E. Coli 4,640,000bp Yeast 12,100,000bp Fruit Fly 140,000,000bp Human 3,000,000,000bp Pea 4,800,000,000bp Wheat 17,000,000,000bp The genomic fragments run at ~12kbp because they are sheared during extraction Analyzing DNA Samples in a Research Lab Using the protocol in the Cereal Genomics module, the genomic DNA extracted will look different than the optimized DNA extraction on the previous slide ( this is mainly due to the missing phenol/chloroform step) This is expected. Even though this genomic DNA preparation is not perfect, it is suitable for use as a PCR template Lane A: Barley Lane B: Corn Lane C: Oat Lane D: Rice Lane E: Wheat Note that the DNA has sheared (particularly for wheat) – broken up into numerous fragments and is not a clean single band at the top – these are the mid-ranged sized fragments (1000-10,000bp size range) The bright bands at the 100 - 1000 bp range are RNA, which also gets extracted using this protocol Analyzing DNA samples in a Classroom Lab Analysis of samples: Barley (A): This sample is fine Corn (B): This sample is fine Oat (C) : This sample is fine Rice (D) : This sample is fine Wheat (E): This sample has severe degradation, can work for PCR but should re-extract
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