University of the West Indies at St. Augustine - BIOC 2169BIOC2169-DNA Lab 2020 -STUDENT LAB-1 (1).

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STUDENT LAB BIOC2169 – Practical Skills in Biochemistry II (2017/18) Lab title: DNA extraction of genomic DNA from fungi, plant, animal tissues using the CTAB method Modified CTAB DNA Extraction ... Protocol (Lefort and Douglas, 1999; Cold Spring Harbor Protocols,http://cshprotocols.cshlp.org/content/2007/11/pdb.ip47.full doi:10.1101/pdb.ip47Cold Spring Harb Protoc 2007). Lab Objectives: 1. To extract DNA from a animal tissues using CTAB method 2. To quantify DNA using spectrophotometric method 3. To determine the purity of the extracted DNA 4. To calculate the concentration of extracted DNA 5. To visualise extracted DNA using agarose gel electrophoresis 1. Tissue Fresh tissue should be collected and used but samples kept at 4°C can be used the next day. For this lab, students will extract DNA from:-  Animal liver, heart and brain 2. Reagents and equipment • Extraction buffer (EB): 50 mM Tris-HCl (pH 8.3), 0.7 M NaCl, 0.4 M LiCl, 20 mM EDTA (pH 8), 1 % (w/v) CTAB, 1 % (w/v) PVP (PVP-40), 2% (w/v) SDS, 1% β-mercaptoethanol (added last and only used for tissue with a high amount of polyphenols e.g. some plant species and seed material).The extraction buffer then also serves as a lysis buffer, which typically consists of the following ingredients in various combinations. • Phenol:chloroform:isoamyl alcohol (25:24:1) • Chloroform-isoamyl alcohol (24:1) • Isopropanol (kept at -20°C) • 70% ethanol(kept at -20°C) • 10mM Tris buffer (pH 8.3) • Acid washed, ethanol/bleached mortar and pestle for larger preps or micropestles for micropreps. • Microcentrifuge tubes (2 ml and 1.5 ml) • Sterile tips and micropipettes • Sterile distilled water • Water bath • Ice in ice buckets • Microcentrifuge • Agarose gel electrophoresis apparatus Dr. Sephra Rampersad 04.01.18 [Show More]

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