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DNA extraction from plants – recap

DNA extraction from plants – recap. Smush (“homogenize”) the cells Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors Remove solids with centrifugation Remove proteins with KOAc precipitation Precipitate DNA with alcohol

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DNA extraction from plants – recap

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  1. DNA extraction from plants – recap • Smush (“homogenize”) the cells • Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors • Remove solids with centrifugation • Remove proteins with KOAc precipitation • Precipitate DNA with alcohol • Remove DNA-bound proteins with phenol-chloroform and chloroform extraction

  2. Waiter! There’s RNA in my food too…

  3. DNA A stable, double helix designed to store information and be chemically unreactive

  4. RNA A different tale T. thermophilus - Ramakrishnan et al., Cell, 2002

  5. RNA extraction from plants • Smush (“homogenize”) the cells • Dissolve membranes and solublize RNA in a detergent buffer with RNAse inhibitors • Remove solids with centrifugation • Remove proteins with phenol extraction • Remove DNA with LiCl precipitation • Precipitate RNA with alcohol

  6. Nucleus (DNA, snRNA, siRNAs) Chloroplast (rRNA, mRNA, tRNA) Vacuole (RNAse!) Mitochondrion (rRNA, mRNA, tRNA) Cytosol (mRNA, tRNA, rRNA) Cell Wall Cell Membrane

  7. RNA extraction issues • Homogenization • RNAse • DNAcontamination • pH of phenol RNAse

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