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DNA extraction from microbial mats

DNA extraction from microbial mats. Alejandro López-Cortés & José Q. García-Maldonado. June, 2011. For looking life on Mars. The search life on other planets starts with the comprehension of life on Earth. Thus, w e must first identify the characteristics of life

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DNA extraction from microbial mats

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  1. DNA extraction from microbial mats Alejandro López-Cortés & José Q. García-Maldonado June, 2011

  2. For looking life on Mars • The search life on other planets starts with the comprehension of life on Earth. Thus, we must first identify the characteristics of life • Characteristics of life such reproduction, heredity, energy obtention, homeostais, adaptation and evolution of cells are codify by a biomolecule named deoxyribonucleic acid or DNA • DNA is a complex molecule (polymer) carrying the information for the development of every trait, or characteristic, of an organism

  3. DNA is compose by nucleotides which consists of a phosphate group, a five-carbon sugar (deoxyribose), and a nitrogenous base (adenine, thymine, cytosine, guanine) Chemistry of DNA

  4. In cells, DNA is presented in double-stranded helix form, which are complementary and antiparallel, the 3' end on one side of the ladder is opposite the 5' end on the other side. Structure of DNA

  5. Geochemistry • DNA or their components could be detect in rocks and sediments of planet Earth • These biosignatures could be search in samples of rock of Mars

  6. Microbial Ecology • In ME is the critical fist step for many subsequent molecular biological techniques (e.g., PCR, cloning and sequencing), having as a final goal the analysis of the composition of the microbial community through their genetic material.

  7. Microbial Mats • Microbial mats are the modern counterparts of stromatolites (fossils laminated rocks built by microorganims) • Our activity, consider the use of microbial mats from Baja California Sur, Mexico to isolate DNA

  8. Steps to recovery DNA • Cell lysis with chemical and mechanical disruption. • Removing membrane lipids, proteins and inhibitors. • Precipitation of DNA with ethanol. • Visualization of DNA in an agarose gel

  9. Non-toxic protocol for DNA Extraction from microbial mats and strawberries Cell lysis • Crush 4 grams of microbial mats or 2 strawberries in a mortar with help of a pestle • Transfer the crushed material (microbial mats or strawberries) into the beaker (250 ml) • Add 25 ml of cold distilled water and mix gently with the spatula by 3 min. • Add a pinch of salt (sodium chloride). • Mix with a spatula for about 5 minutes until obtain a homogeneous solution

  10. Non-toxic protocol for DNA Extraction from microbial mats and strawberries Removing membrane lipids, proteins and inhibitors • Add 15 ml of liquid soap (“Axion”) and mix for 15 minutes. • Add 0.1 g of papaine (crude powder Sigma P3375). • Mix for 5 minutes or until obtain a homogeneous solution.

  11. Non-toxic protocol for DNA Extraction from microbial mats and strawberries Precipitation of DNA • Tranfer to a glass tube using a funnel to try to do not touch the wall of the tube. Fill only 1/3 of the glass tube • Slowly add cold ethanol through the wall of test tube until 90% of their capacity and maintain in a slant position the tube • Wait until the white-yellow filaments are going up or floating • With a Pasteur pipettes with a curved tip take the filaments and air dry for 5 min • Transfer the dried filaments into the plastic 1.5 ml vial, containing 50µl of loading buffer • Incubate for about 30 minutes at room temperature

  12. Non-toxic protocol for DNA Extraction from microbial mats and strawberries Electrophoresis (visualization of DNA) • Using a microwave, dissolve 0.8 g of agarose in 80ml of TBE buffer • Pouring the melted agarose in the tray with the combs • Wait until gelification • Load the sample in the well • Run for 30 minutes at 100Volts • Take out the gel and transfer to the plastic container • Add 500 ml of Fast Blast solution 100X • Incubate in a dark for 5 minutes • Take out the gel and transfer to another plastic container with tap water • Shake gently for about 5 minutes, discard the water and repeat the step 23. • After 5 minutes the DNA bands will appear • Dry the gel and take a picture.

  13. Materials Beaker 250 ml Cylinder (50 ml) Mortars and pestles Spatula Test tubes Funnel Pasteur pipettes with a curved tip Plastic vials of 1.5 ml Micropipette (10-100 µl) Plastic tips Electrophoresis chamber Power supply Plastic container for the staining Nitrile gloves Reagents 4 g of fresh microbial mats or 2 frozen strawberries 25 ml of cold distilled water A pinch of salt (sodium chloride) 15 ml of liquid soap (“Axion”) 0.1 g of papaine (crude powder Sigma P3375) 20 ml of cold ethanol Agarose Fast Blast reagent from BIORAD Loading buffer (Blue/Orange Loading dye 6x Promega) Trizma boric acid EDTA (TBE buffer) Non-toxic protocol for DNA Extraction from microbial mats and strawberries

  14. Thanks for your attention!!

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