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Dna Extraction

It's about DNA extraction, isolation and culturing.

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15 views16 pages

Dna Extraction

It's about DNA extraction, isolation and culturing.

Uploaded by

vngeke
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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TEADY BIOSCIENCE

DNA EXTRACTION …………………

OLUSOLA ELEKOFEHINTI
30 MAY, 2023

presented during Basic Training and Molecular Biology and Bioinformatics


Techniques for SLT students of Ekiti State University, Ado Ekiti
Chromosomal and Plasmid DNA
❖Chromosomal DNA is much bigger than plasmid DNA

❖In bacterial and eukaryotes, often circular


chromosomes

❖Plasmid is a small, circular, double-stranded DNA


molecule that is distinct from a cell's chromosomal
DNA.

Bacterium Animal cell


Plant cell
Sources of DNA
1. Bacteria
2. Blood
3. Buccal cells
4. Cultured cells
5. Biopsies
6. Forensic samples(like body fluids, bone, teeth roots
and hair follicle)

Bacterium Cultured cells Buccal cells collection Blood


Basic rules in DNA isolation
1. Work on ice- this will help to slow down enzymatic
processes
2. Wear gloves to protect your samples from you
(external or exogenous DNA)
3. Use sterilized, DNAse and RNAse free solutions
and disposables
4. Autoclave all solutions and store in fridge
(except SDS and organic solvents)
Basic Steps in DNA Isolation
❖DNA isolation is a routine laboratory procedure to
collect DNA for further molecular analysis

❖It generally involves three basic stages:

1. Cell disruption: disruption of the cellular structure


to create a lysate. This is commonly achieved by

i. Grinding, sonicating or by physical shearing of the


Sample

ii. Removing membrane lipids by adding a detergent or


chaotropic salts
2. Isolation of DNA:
i. separation of the soluble DNA from cell debris
and other insoluble material, and
ii. Removal of proteins

3. Precipitation of DNA:
i. adding ice-cold ethanol or isopropanol to
aggregate the DNA together
ii. Centrifuging to form a DNA pellet
This step also remove alcoholic soluble salt
Cell culture/ Cell Lysis
Tissue extract Physical/Chemical
Wash steps Detergent

Removal of membrane lipids


Wash steps Protease treatment

Protein denaturation and removal


Wash steps

Removal of other cellular contaminants


RNAse treatment

RNA removal
Elution in slightly alkaline buffer/
RNAse free water
Purified DNA
DNA Quantification
Quantification of DNA
DNA concentration, yield and purity can be assessed
using three different methods:

1. Absorbance

2. Agarose gel electrophoresis and

3. Fluorescent DNA-binding dyes.


Absorbance method
❖The most common technique to determine DNA
yield and purity is also the easiest method

❖All that is needed for measurement is a


i. spectrophotometer equipped with a UV lamp,
ii. UV-transparent quarzt cuvettes and
iii. a solution of purified DNA

NanoDrop
❖Absorbance readings are performed at 260nm (A260)
where DNA absorbs light most strongly

❖To ensure the numbers are useful, the A260 reading


should be between 0.1–1.0.
Calculations using absorbance value
❖The concentration of DNA (µg/ml) can be obtained
using the formula:

Concentration (µg/ml) =A260 reading × df × 50µg/ml

Where, df = dilution factor


A260 of 1.0 = 50µg/ml pure dsDNA
A260 of 1.0 = 33µg/ml pure ssDNA
A260 of 1.0 = 44µg/ml pure RNANA

Amount of DNA (Yield) (µg) =


DNA Concentration (µg/ml) × Total Sample Volume (ml)
DNA Purity
❖Since RNA also has a great absorbance at 260nm

❖The presence of guanidine will lead to higher 260nm


absorbance

❖The aromatic amino acids present in protein absorb


at 280nm

❖The present of all these in the DNA solution, will


contribute to the total measurement at 260nm
DNA Purity
❖The most common purity calculation is determining
the ratio of the absorbance at 260nm divided by the
reading at 280nm

❖ Good-quality DNA will have an A260/A280 ratio of


1.7–2.0.

‹ 1.7 = protein contaminated


› 2.0 = chloroform/phenol contaminated
Factors to consider in primer design

Length of primers of the primer (18-24)

Annealing temprature and melting temprature of


the primer

G-C content of the primer

Secondary structure of the primer

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