BT 403+Mod III+NKJ+Lecture+ 2 DNA+Isolation 2011

7/28/2019 BT 403+Mod III+NKJ+Lecture+ 2 DNA+Isolation 2011

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BT-403 MOD-III NKJ LECTURE : ISOLATION OF DNA

Whenever the quantity of DNA that you need to work with can not be obtained directly

or through PCR, or you need a constant source of identical DNA, cloning of the DNA isthe solution.

SOURCES OF DNA

There are four primary sources of DNA: Cells, Organelles, Plasmids, and Viruses.

There are separate considerations for the isolation of DNA from each of these.

Each of the DNA sources must be purified away from proteins, RNA, carbohydrates

and lipids.

Sometimes DNAs must be purified away from each other. For example, in isolating

organellar DNA it is easier to first isolate the organelle separate from the nuclei and thenextract the organellar DNA. In other cases, such as in plasmid isolstion from bacteria, the

chromosomal DNA and plasmid DNA reside in the same cell and must be separated byphysical differences between the two.

CONCENTRATING CELLS

Centrifugation is a method that allow cells to be concentrated and separated from the

media in which they are grown. Many media are complex and so this provides a goodfirst step in removing potential contaminants.

DNA PURIFICATION FROM CELLS


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Once cells are concentrated and separated from growth media, the cells can be lysed.

There are various way to lyse cells.

Source Lysis Methods

Bacteria

Lysozyme - degrades polysaccharide cell walls

SDS - detergent for cell membranes

Freeze/Thaw several timesFrench Press

Organelles

OsmoticSonication

Detergent

Animal Cells

Osmotic

Sonication

Detergent

Plant Cells

Several enzymes to break down cell walls

Mechanical grinding

REMOVING CONTAMINANTS

Lysis and centrifugation to remove cell debris.

Contaminant Method of Removal

Cell Walls & Cellular Debris Centrifugation

Carbohydrates CTAB + NaCl


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Protein

Phenol/CHCl3 extractionProtease

Guanidinium Thiocyanate & Silica

CsCl gradient with ethidium bromide

RNA

RNase


CTAB extraction

Phenol extraction


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Glass milk purification of DNA using ITC.

CONCENTRATING DNA AND CHANGING SOLVENTS

Ethanol precipitation (70% + salt to neutalize negative charges)

Also precipitates RNA and some protein.


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Precipitation of DNA with ethanol.

Reduce aqueous volume by extracting with butanol. Butanol excludes DNA and

absorbs water.

PLASMID ISOLATION

Plasmid DNA must be isolated free from chromosomal DNA.

There have been a number of methods described over the years but the most popular

and commonly used method is the Alkaline Lysis method.

1- Pellet cells by centrifugation, discard supernatant.

2- Resuspend in Solution I (cloudy suspension, no clumps)

Glucose - osmoticantTris - buffer

EDTA - chelated divalent metal cations (inhibits RNases and DNases)

Lysozyme - optional, breaks down cell wall polysaccharide

3- Mix gently with Solution II (clear, viscous)

SDS - Detergent to lyse cell wallsNaOH - raises pH to over 11.3 => denatures DNA

4- Mix gently with Solution III (white clumps, like small curd cottage cheese)

3M KAc - lowers pH, neutralizes DNA which renatures quickly in the high salt.

Causes the complementary single strands to zip together quickly while the genomic DNAform an insoluble glob because it can't find the right complementary strand.


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5- Centrifuge to pellet the cellular debris and the insoluble genomic DNA mass.

6- Ethanol precipitate to change the solvent.

7- Resuspend in a buffer. (still contains some RNA and Protein as contaminants)

FURTHER PURIFICATION

Various column types, Qiagen, Promega, ...



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HOW TO DETERMINE THE QUANTITY AND QUALITY OF YOUR DNA

The quality of DNA can be determined from a wavelength scan between 260 and 320nm. DNA and proteins do not absorb at 320 nm and any significant readings usually

come from the light scatter of insoluble particles, such as denatured proteins. Proteins

absorb at a maximum around 280 nm, while nucleic acids, such as DNA and RNA, showmaximal absorption at 260 nm.

The quality of DNA is often initially estimated by looking at the ratio of the absorbance

at 260 nm / 280 nm.

A260 / A280 Quality

Ratio < 1.8 Contains protein contamination

1.8 < Ratio < 1.9 no contamination

Ratio > 2 DNA may contain RNA


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Absorbance peak or shoulder at 270 nm Phenol contamination

The quantity of pure nucleic acids can be estimated from the following

Nucleic Acid Form NA Concentration at 1 A260Double -stranded DNA 50 g/ml

Single-stranded DNA 37 g/ml

RNA 40 g/ml

BT 403+Mod III+NKJ+Lecture+ 2 DNA+Isolation 2011

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