Advances in Molecular Techniques

Rakesh S. Sengar, Amit Kumar, Reshu Chaudhary, Ashu Singh

• Publisher: CRC Press
• Year: 2018
• Print ISBN: 9780815370758
• eBook ISBN: 9781351021203
• Topic: Medicine
• Subtopic: Biotechnology in Medicine

Molecular genetics aims to comprehend biological activity at the gene sub-level. Scientists from different areas of research and applied science can use the standard techniques optimized by molecular biologists.This book serves as a guide that introduces classic molecular biology techniques and advances in molecular and genetic engineering.

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DNA Manipulative and Ingratiative Techniques

Isolation of Total Genomic DNA

Isolation of DNA from different tissues, either plant or plasmid, is at the heart of molecular biology because all the genetic information is carried in the linear sequence of nucleotides in DNA. Several protocol have been developed for the isolation of genomic DNA, but selective protocol are described below for plant, plasmid, yeast, and bacteria.

Isolation of Plant DNA Using the CTAB Method

Introduction

Isolation of DNA from plant tissues is at the heart of plant molecular biology. Because plant cells are surrounded by rigid cell walls and because plant tissues often contain a variety of secondary metabolites that can damage DNA, isolation from plants can present some particular difficulties. Among the many protocols developed for DNA isolation from plants, the method presented here is one of the simplest and most effective for a variety of plant species.

DNA isolated by methods such as the one presented here represents total cellular DNA. In plants, this means the isolation of 3 distinct genomes:

1.The nuclear genome

2.The chloroplast genome

3.The mitochondrial genome

Generally, when we talk about plant DNA, we mean nuclear DNA, but it is important to remember that chloroplasts and mitochondria each have distinct genomes. These genomes, like the nuclear genome, are targets of research in molecular biology and genetic engineering. Total plant DNA isolated here will be compared with DNA isolated from chloroplasts.

Experimental Outline

Day 1: DNA isolation (2 h).

Grind tissue and suspend in extraction buffer.

(EB) incubate at 65°C for 1 h.

Extract with chloroform.

Precipitate DNA with isopropyl alcohol.

Resuspend the DNA in buffer and store.

Day 2: Compare this DNA preparation with DNA isolated from chloroplasts by gel electrophoresis.

Materials Required

Chemicals

•For EB (extraction buffer): 50 mM Tris (pH 8.0), 1% CTAB, 50 mM EDTA, 1 Mm 1-10-o-phenanthroline, 0.7 M NaCl, 1% beta-mercaptoethanol

•Chloroform

•Isopropyl alcohol

•80% ethanol

•15 mL ammonium acetate

•For TE buffer: 10 mL Tris (pH 8.0), 1 mL EDTA (pH7.5)

Glassware and Other

•Measuring bottle

•Conical flask

•Measuring cylinder

•Mortar and pestle

•Centrifuge tubes (50 mL capacity, capped)

Equipment

•Water Bath

•Cooling Centrifuge

•Gel doc system

•Electrophoresis unit

•Power pack

•Micropipette

Pre-Lab Preparation

Pea tissue used in this protocol should be air-dried or freeze-dried. Grow the plants either in sand or perillite. Tissue can be air-dried in the laboratory by spreading the cut shoots on a sheet of absorbent paper and turning daily. Tissue treated in this way will typically dry in 5–7 days. Dried tissue can be saved for use at a later date by sealing in a tightly closed jar and freezing.

Method (Figure 1.1)

Da...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Dedication
• Contents
• Foreword
• Preface
• Authors
• Chapter 1: DNA Manipulative and Ingratiative Techniques
• Chapter 2: Regulative Techniques
• Chapter 3: Recombinant Techniques
• Chapter 4: Nanobiotechnology and Metabolome Profiling
• Appendix
• Glossary
• Bibliography
• Index