Laboratory: Unit 3: prepare genomic DNA (53-54) Lecture: Genomic DNA purification In-Class Writing: discuss JBC 266: 3811-14 (1991) (page 155) Hand In:

Slides:

Advertisements

Similar presentations

Molecular Genetic.

Advertisements

Laboratory: Unit 3: isolate bacteria (pages 43-52) Unit 4: extract DNA (pages 71-83) Next Day: examine plates & streak Lecture: DNA extraction from bacterial.

A Quick Overview of Lemaux Lab’s DNA Extraction Protocol Eric Trieu March 2009.

IMMUNOLOGY LABORATORY PBMC ISOLATION SOP by Kizza D Martin Ssemambo

DNA Extraction: A User’s Manual Margarita Hernandez Instruction Manual ENC /28/14 Margarita Hernandez Instruction Manual ENC /28/14 A step.

Isolation and Quantification of Nucleic Acids in Plants

Materials CTAB buffer Microfuge tubes Mortar and Pestle Microfuge Absolute Ethanol (ice cold) 70 % Ethanol (ice cold) 7.5 M Ammonium Acetate 55o C water.

DNA, Chromosomes By Dr. : Naglaa Mokhtar. DNA Structure.

Plasmid Minipreps Kits….

Extraction of Nucleic Acids (Genomic DNA, mRNA and Plasmid DNA)

Organic Extraction Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Cloning a DNA segment from bacteriophage Recombinant DNA transformed into bacterial cells Last week we plated cells onto agar plates + ampicillin + X-gal.

Cloning a DNA segment from sheep Recombinant DNA transformed into bacterial cells Last week we plated cells onto agar plates + ampicillin + X-gal Controls:

Lab 6 Isolation Techniques

Plasmid DNA Isolation Exercise 8.

Mini-Prep Plasmid Isolation and Identification. Page 3-53 in lab manual & handout.

General Genetics. 1. Be introduced to the laboratory techniques involved in DNA extraction. 2. Test DNA integrity using gel electrophoresis.

 DNA extraction is a procedure used to isolate large amounts of DNA from cell.  DNA can be isolated from plant and animal cells as well as from bacteria.

Modified Method for Combined DNA and RNA Isolation From peanut and Other Oil Seeds Phat M. Dang and Charles Y. Chen USDA-ARS, National Peanut Research.

DNA Extraction Margarita Hernandez Instruction Manual ENC /28/14 Margarita Hernandez Instruction Manual ENC /28/14 A step by step guide on.

Mini-Prep Plasmid Isolation and Identification. Page 3-53 in lab manual & handout.

Chelex ® Extraction. Learning Objectives Competence in extraction of different biological stains. Knowledge of the theory of DNA Isolation using Chelex.

Cat # SL Store at 4~23 0 C DiatoCLEAN™ DNA Purification Kit Quick Protocol Small 300 Preps Large 600 Preps Gaither Drive Gaithersburg,

BIOLOGY 3020 Fall 2008 “Keys of Corn” Project Plasmid isolation Genetic Diversity in corn. Lots of different types of corn are offered for sale at the.

Important points on DNA isolation

Extraction and Quantitation of DNA From E. coli

Proteomics Module Day 1 Tech talk. Experiment: Yeast protein expression changes caused by H 2 O 2 exposure. ► 2 Control groups (A and B): nothing added.

Proteomics Module Day 1 Tech talk 10 students in 5 groups of 2.

CAVEAT!!! Usually we do these procedures with a pure culture ◦ Grow bacteria in broth ◦ Streak on plate ◦ Grow single colony on broth.

RNA Extraction.

5. Internet Exploration:

Isolation and Purification of DNA from Escherichia coli GROUP 2 Chester Mancia Frances Miclat Mark Mosses Oliva HUB 42.

Introduction to Vectors In order to study a DNA fragment (e.g., a gene), it needs to be amplified and eventually purified. These tasks are accomplished.

To perform in situ studies on mRNA or DNA, cells morphology and genetic information has to be preserved. Fixation(preservation) is conducted with 3.4.

CELL ENVELOPE PREPARATION / SOLUBILIZATION Kris-itd.unair.ac.id (for education purpose only)

CAPE Biology Workshop on Concepts in Biotechnology & Genetic Engineering Prepared and presented by Dr. Marcia E. Roye.

Isolation of biological macromolecule Technology to simply go into a mixture and grab a single type of molecule is not readily available Instead use procedures.

DNA Isolation Lab.

DNA extraction.

DNA Extraction from fruit Lab.2 Alanoud Alfaghom.

Isolation of Bacteriophages

Manual Extraction of DNA from The Blood. - Blood Sample. - Distilled water. Dionized water. - Ice and Plastic bucket.- Materials.

Lab 5 DNA Extraction.

Manual Extraction of DNA from The Blood. Materials - Blood Sample. - Distilled water. Dionized water. - Ice and Plastic bucket.-

1. Be introduced to the laboratory techniques involved in DNA extraction. 2. Test DNA integrity using gel electrophoresis.

MISS :Salsabeel AL Jou jou

Lecturer: Bahiya Osrah DNA ISOLATION. DEOXYRIBONUCLEIC ACID OR DNA The molecule that controls everything that happens in the cell. DNA contains the genetic.

DNA extraction.

RNA isolation from monolayer cell Vascular Genomics Laboratory

PLASMID ISOLATION AND ANALYSIS Part III Plasmid Isolation.

Extraction of Human DNA from blood

DNA extraction. Total DNA: whole blood (fresh or frozen), plasma, serum, buffy coat, body fluids, lymphocytes and cultured cells. This technology first.

DNA Extraction from human blood

Extraction of Human DNA

Isolation of DNA Biotechnology.

TYPES OF ISOLATION.

Mini-Prep Plasmid Isolation and Identification

Lab no. 10 Plasmid DNA isolation.

DNA Isolation from Haman Blood Cells

The common lysis solutions contain A. sodium chloride.

DNA EXTRACTION Protocol and notes 9/17/2018.

Mini-Prep Plasmid Isolation and Identification

DNA Extraction from Blood

Lab no. 10 Plasmid DNA isolation.

The cell The basic unit of any living organism.

Presentation transcript:

Laboratory: Unit 3: prepare genomic DNA (53-54) Lecture: Genomic DNA purification In-Class Writing: discuss JBC 266: (1991) (page 155) Hand In: flow chart 3; lab report 2 Read: AEM 63: , 1997 (minus abstract) (see page 68) Due Next Class: abstract for AEM 63:

Use one isolate. Pick a dense culture. Transfer 1 ml culture to sterile vial ml DMSO; shake to mix. Label with your seat number. Wrap tape around vial so ends overlap. Place in freezer box. Place remaining culture in 1.5-ml tube; purify genomic DNA (section D).

Purify Genomic DNA 1. Transfer 1.5 ml culture to centrifuge tube. 2. Centrifuge maximum speed 1 minute. Discard supernatant (autoclave). 3. Suspend cells in 450  l of Tris + EDTA; disperse pellet completely. EDTA chelates Mg ++  inhibits nucleases & helps disrupt cells.

4. Add 20  l lysozyme, 20 min, 37 o C. Lysozyme cleaves peptidoglycan cell wall. 5. Add 10  l proteinase K, 20 min, 50 o C. Proteinase K cleaves cell wall proteins & nucleases.

6. Add 20  l 25% SDS, 10 min, 68 o C. SDS = ionic detergent; disrupts membrane lipids & denatures proteins. 7. Add 57  l 5 M NaCl; vortex 1 minute. DNA & RNA precipitate in high salt/ethanol.

8. Incubate 5 min, 68 o C; vortex 1 minute. Shears DNA; reduces viscosity. 9. Add 0.5 ml phenol:chloroform:isoamyl alcohol (25:24:1) equilibrated with 1 M Tris. Mix well. Avoid contact with organic solvents; work in fume hood. Lab coats, gloves, goggles, closed shoes required. If phenol contacts skin, wash with water & seek medical attention. 10. Centrifuge maximum speed, 5 minutes.

11. Transfer aqueous (top) phase to 1.5-ml tube with 0.5 ml chloroform:isoamyl alcohol (24:1). Withdraw top phase; leave cell debris at interphase. DNA attached to membrane will tug at interphase. Discard organic solvents in organic waste. 12. Mix well; centrifuge maximum speed, 2 min.

13. Transfer aqueous (top) phase to 1.5-ml tube. Leave cell debris at interphase. Discard organic solvents in organic waste. 14. Add 1 ml ethanol (ice cold), mix thoroughly; hold 15 min, 0 o C or 4 o C. 15. Centrifuge maximum speed, 5 min; orient tube in rotor so pellet forms at known location. Discard supernatant solution.

16. Wash DNA/RNA pellet with 0.5 ml 70% ethanol. Let stand 1 minute; do not disturb pellet. 17. Centrifuge maximum speed, 2 minutes. Discard supernatant solution carefully; pellet will not adhere tightly to tube.

18. Centrifuge 10 seconds. Remove ethanol with pipet tip. Air dry pellet. 19. Dissolve DNA/RNA pellet in 50  l DNA buffer. Freeze at -20 o C.