Department of Biochemistry and Molecular Biology

University of Dhaka

MS Practical

Isolation And Purification of Bacterial DNA

Submitted by

Md. Atai rabby

MS

Data

Absorbance at 260nm : 0.161

Absorbance at 280nm : 0.090

Claculation

Ratio of Absorbance = 𝐴260

𝐴280 =

0.161

0.090 = 1.786 ≈ 1.8

1 OD equivalent amount of DNA = 50 µg

0.161 OD equivalent amount of DNA = 50×0.161

= 8.05 µg

Dilution factor = 10×2 = 20

Total amount of DNA = 20 ×8.05 = 161 µg

Result

Total amount of bacterial DNA in 2 ml sample was 161 µg

Ovservation

Three bands were found in Gel electrophoresis indicating

1. Nick,linear chromosomal DNA

2. Compact Chromosomal DNA

3. Plasmid DNA

The ratio of DNA was approximately 1.8 indicating almost pure DNA.

Discussion

The purpose of this experiment was to isolate chromosomal DNA of Bacteria.

Rationale for each step :-

a. Cells must be resuspended in buffer (some protocols call for washing the cells in the buffer---i.e.

resuspending and centrifuging---2 or 3 times) in order to have the ionic strength of the solution compatible

with biomolecules, in particular, the salt and pH. The EDTA is a chelating agent that ties up divalent metal

ions; these ions are often cofactors necessary for the action of DNAses---enzymes present in the cell, which,

when released by lysis along with the DNA, can degrade the DNA.

b. SDS is an ionic detergent which will lyse most cells and denature some proteins.

c. Chloroform is used to deproteinize . The chloroform causes surface denaturation of proteins; the isoamyl

alcohol reduces foaming, aids separation and maintains the stability of the layer of the centrifuged,

deproteinized solution.

d. DNA must become rehydrated completely before it will form a uniform suspension. This can take a long

time, depending on the concentration of the DNA. It can be facilitated by starting off with low ionic strength

buffer and, once resuspended, adding an appropriate amount of concentrated buffer stock to bring the buffer

to the correct ionic strength.

e. Proteinase K is a ubiquitous protein-degrading enzyme. It can function in the presence of detergent and at

elevated temperatures. The elevated temperatures are used to denature DNAses and to facilitate DNA-

protein dissociation.

f. Ethanol lowers the effective water concentration, causing large biomolecules to interpenetrate and

aggregate. The result is a visible precipitate at the interface, where the ethanol is concentrated. As DNA is

precipitated and removed, more is exposed to the ethanol and will precipitate.

Phenol-chloroform extraction (abbreviated PC or PCIA, see reagents below) is a liquid-liquid

extraction technique in biochemistry. It is widely used in molecular biology for isolating DNA,

RNA and protein. Equal volumes of a phenol:chloroform mixture and an aqueous sample are

mixed, forming a biphasic mixture. This method may take longer than a column-based system such

as the silica-based purification, but has higher purity and the advantage of high recovery of RNA.

Phenol: The phenol used for biochemistry comes as a water-saturated solution with Tris

buffer, as a Tris-buffered 50% phenol, 50% chloroform solution, or as a Tris-buffered 50%

phenol, 48% chloroform, 2% isoamyl alcohol solution (sometimes called "25:24:1"). Phenol

is naturally somewhat water-soluble, and gives a fuzzy interface, which is sharpened by the

presence of chloroform, and the isoamyl alcohol reduces foaming. Most solutions also have

an antioxidant, as oxidized phenol damages the nucleic acids. For RNA purification, the pH

is kept around pH 4, which retains RNA in the aqueous phase preferentially. For DNA

purification, the pH is usually near 7, at which point all nucleic acids are found in the

aqueous phase.

Chloroform: Chloroform is stabilized with small quantities of amylene or ethanol, because

exposure of pure chloroform to oxygen and ultraviolet light produces phosgene gas. Some

chloroform solutions come as pre-made a 96% chloroform, 4% isoamyl alcohol mixtures that

can be mixed with an equal volume of phenol to obtain the 25:24:1 solution.

ADDITIONAL NOTES:

The isolation of DNA is one of the more commonly used procedures in many areas of bacterial

physiology, genetics, molecular biology and biochemistry. Purified DNA is required for many

applications such as studying DNA structure and chemistry, examining DNA-protein interactions,

carrying out DNA hyrbridizations, sequencing or PCR, performing various genetic studies or gene

cloning. The isolation of DNA from bacteria is a relatively simple process. The organism to be used

should be grown in a favorable medium at an optimal temperature, and should be harvested in late

log to early stationary phase for maximum yield. The cells can then be lysed and the DNA isolated

by one of several methods. The method of choice depends in part on the organism of interest and

what the DNA will be used for after purification. Following lysis, other cellular constituents are

selectively removed. Once this is accomplished, DNA can be precipitated from solution with

alcohol and dissolved in an appropriated buffer.

The lysis of the bacteria is initiated by resuspending a bacterial pellet in a buffer containing

lysozyme and EDTA. In addition to inhibiting DNAses, the EDTA disrupts the outer membrane of

the gram-negative envelope by removing the Mg++ from the lipopolysaccharide layer. This allows

the lysozyme access to the peptidoglycan. After partial disruption of the peptidoglycan, a detergent

such as SDS is added to lyse the cells. Most gram-negative cells will lyse after this treatment and

many can even be lysed without lysozyme. Once the cells are lysed, the solution should be treated

gently to prevent breakage of the DNA strands.

Subsequent steps involve the separation of the DNA from other macromolecules in the lysate. Both

phenol (that has been equilibrated with Tris buffer) and chloroform (with isoamyl alcohol as a

defoaming agent) are commonly used to dissociate protein from nucleic acids. These reagents also

remove lipids and some polysaccharides. Proteolytic enzymes such as pronase or Proteinase K are

often added to further remove protein. Proteinase K is a particularly useful enzyme in that it is not

denatured by SDS and in fact works more effectively in the presence of SDS. The nucleic acids

(including RNA) may then be precipitated in ice cold ethanol if the ionic strength of the solution is

high. This is followed by RNAse treatment to degrade the RNA. The solution may then be

reprecipitated with ethanol. In this precipitation, the ribonucleotides from RNase treatment will

remain in solution leaving purified DNA in the pellet. The pellet can then be dissolved in an

appropriate buffer.

Alcohol precipitations of DNA and RNA are widely used in molecular biology and are valuable

because they allow the nucleic acids to be concentrated by removing them from solution as an

insoluble pellet. If concentrations of DNA are relatively high (>1ug/ml) DNA can be effectively

precipitated in 10-15 min by shielding the negative charge with monovalent cations (0.3M Na or 2.5

M ammonium ions are commonly used) followed by the addition of 2 volumes of 95% ethanol.

Factors affecting alcohol precipitations are given below.

A major consideration in any DNA isolation procedure is the inhibition or inactivation of DNases

which can hydrolyze DNA. The buffer in which the cells are suspended should have a high pH (8.0

or greater) which is above the optimum of most DNases. EDTA is also included in the resuspension

buffer to chelate divalent cations (such as Mg++) which are required by DNases. The SDS also

reduces DNase activity by denaturing these enzymes. DNase activity is further controlled by

keeping cells and reagents cold, using proteolytic enzymes such as pronase or proteinase K, and a

heating step that will thermally denature DNase (but must not be hot enough to denature the DNA)

The procedure used here is useful for isolating DNA from a large variety of gram negative bacteria.

It yields partially purified DNA of sufficient quality for most techniques, such as restriction

digestion, ligation , and cloning. Further purification by additional solvent extraction may be

required for experiments needing purer DNA (e.g. physical chemical studies such as melting curves,

etc.)

Agarose gel electrophoresis

Agarose gel electrophoresis is a method used in clinical chemistry to separate proteins by charge

and or size (IEF agarose, essentially size independent) and in biochemistry and molecular biology

to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA

and RNA fragments or to separate proteins by charge. Nucleic acid molecules are separated by

applying an electric field to move the negatively charged molecules through an agarose matrix.

Shorter molecules move faster and migrate farther than longer ones because shorter molecules

migrate more easily through the pores of the gel. This phenomenon is called sieving. Proteins are

separated by charge in agarose because the pores of the gel are too large to sieve proteins.

Agarose gels are easily cast and handled compared to other matrices and nucleic acids are not

chemically altered during electrophoresis. Samples are also easily recovered. After the experiment

is finished, the resulting gel can be stored in a plastic bag in a refrigerator.

There are limits to electrophoretic techniques. Since passing current through a gel causes heating,

gels may melt during electrophoresis. Electrophoresis is performed in buffer solutions to reduce pH

changes due to the electric field, which is important because the charge of DNA and RNA depends

on pH, but running for too long can exhaust the buffering capacity of the solution. Further, different

preparations of genetic material may not migrate consistently with each other, for morphological or

other reasons.

Factors affecting migration of nucleic acids :The most important factor is the length of the DNA

molecule, smaller molecules travel faster, except in field inversion , where it is possible to have

"band inversion" - large molecules travel faster then small molecules.. But conformation of the

DNA molecule, such as % single strand, supercoiling, etc, is also a factor. When analyzing

molecules by size, it is most convenient to analyze only linear molecules to avoid this problem, eg

DNA fragments from a restriction digest, linear DNA PCR products, or RNAs. Agarose gel

electrophoresis is widely used to resolve circular DNA with different supercoiling topology, and to

resolve fragments that differ due to DNA synthesis (Fangman work).

DNA damage due to increased cross-linking will dose-dependently reduce electrophoretic DNA

migration.

Increasing the agarose concentration of a gel reduces the migration speed and enables separation of

smaller DNA molecules. The higher the voltage, the faster the DNA moves. But voltage is limited

by the fact that it heats the gel and ultimately causes it to melt. High voltages also decrease the

resolution (above about 5 to 8 V/cm).

Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with

different speeds (slowest to fastest: nicked or open circular, linear, or supercoiled plasmid).

Visualization by ethidium bromide (EtBr) and dyes : The most common dye used to make DNA

or RNA bands visible for agarose gel electrophoresis is ethidium bromide, usually abbreviated as

EtBr. It fluoresces under UV light when intercalated into DNA (or RNA). By running DNA through

an EtBr-treated gel and visualizing it with UV light, any band containing more than ~20 ng DNA

becomes distinctly visible. EtBr is a known mutagen, and safer alternatives are available.

Even short exposure of nucleic acids to UV light causes significant damage to the sample. UV

damage to the sample will reduce the efficiency of subsequent manipulation of the sample, such as

ligation and cloning. If the DNA is to be used after separation on the agarose gel, it is best to avoid

exposure to UV light by using a blue light excitation source such as the Xcitablue UV to blue light

conversion screen from Bio-Rad or Dark Reader from Clare Chemicals. A blue excitable stain is

required, such as one of the SYBR Green or GelGreen stains.

Blue light is also better for visualization since it is safer than UV (eye-protection is not such a

critical requirement) and passes through transparent plastic and glass. This means that the staining

will be brighter even if the excitation light goes through glass or plastic gel platforms.

SYBR Green I is another dsDNA stain, produced by Invitrogen. It is more expensive, but 25 times

more sensitive, and possibly safer than EtBr, though there is no data addressing its mutagenicity or

toxicity in humans. SYBR Safe is a variant of SYBR Green that has been shown to have low

enough levels of mutagenicity and toxicity to be deemed nonhazardous waste under U.S. Federal

regulations. It has similar sensitivity levels to EtBr, but, like SYBR Green, is significantly more

expensive. In countries where safe disposal of hazardous waste is mandatory, the costs of EtBr

disposal can easily outstrip the initial price difference, however.

Since EtBr stained DNA is not visible in natural light, scientists mix DNA with negatively charged

loading buffers before adding the mixture to the gel. Loading buffers are useful because they are

visible in natural light (as opposed to UV light for EtBr stained DNA), and they co-sediment with

DNA (meaning they move at the same speed as DNA of a certain length). Xylene cyanol and

Bromophenol blue are common dyes found in loading buffers; they run about the same speed as

DNA fragments that are 5000 bp and 300 bp in length respectively, but the precise position varies

with percentage of the gel. Other less frequently used progress markers are Cresol Red and Orange

G which run at about 125 bp and 50 bp, respectively.

Visualization can also be achieved by transferring DNA to a nitrocellulose membrane followed by

exposure to a hybridization probe. This process is termed Southern blotting.

Percent agarose and resolution limits :Agarose gel electrophoresis can be used for the separation

of DNA fragments ranging from 50 base pair to several megabases (millions of bases) using

specialized apparatus. The distance between DNA bands of a given length is determined by the

percent agarose in the gel. The disadvantage of higher concentrations is the long run times

(sometimes days). Instead high percentage agarose gels should be run with a pulsed field

electrophoresis (PFE), or field inversion electrophoresis.

Most agarose gels are made with between 0.7% (good separation or resolution of large 5–10kb

DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose dissolved in

electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but a vertical

polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may

break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1%

gels are common for many applications.

Buffers : There are a number of buffers used for agarose electrophoresis. The most common being,

for nucleic acids Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA (TBE). Many other buffers have

been proposed eg,lithium borate, which is almost never used, based on pubmed citatins (LB), iso

electric histidine, pK matched goods buffers, etc; in most cases the purported rationale is lower

current (less heat) and or matched ion mobilities, which leads to longer buffer life. Borate is

problematic; Borate can polymerize, and/or interact with cis diols such as those found in RNA.

TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means

a lower voltage and more time, but a better product. LB is relatively new and is ineffective in

resolving fragments larger than 5 kbp; However, with its low conductivity, a much higher voltage

could be used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis. As

low as one base pair size difference could be resolved in 3 % agarose gel with an extremely low

conductivity medium (1 mM Lithium borate).