Biochemistry practical lesson 3 Isolation nd characterisation of DNA and
proteins
- 1 - DNA
3. ISOLATION AND CHARACTERISATION OF DNA
a) isolation of plasmid DNA on columns (3.1)
b) measurement of DNA concentration by spectrophotometry (3.2)
c) electrophoretic separation of plasmid DNA on agarose gel (3.3)
3.1 ISOLATION OF PLASMID DNA
OBJECTIVE: ISOLATION OF PLASMID DNA FROM E.COLI
TOPICS:
DNA, chromatography, UV spectroscopy
Plasmid DNA: In microbiology and genetics,
a plasmid is a DNA molecule that is separate
from, and can replicated independently of, the
chromosomal DNA. They are doublestranded
and, in many cases, circular. Plasmids
usually occur naturally in bacteria, but are sometimes found in
eukaryotic organisms (e.g., the 2-micrometre ring in Saccharomyces
cerevisiae). Plasmid sizes vary from 1 to over 1,000 kbp.
Isolation of plasmid DNA from E.
coli is a common routine in research
laboratories. You will perform a
widely-practiced procedure that
involves alkaline lysis of cells and
chromatography on DEAE column.
This protocol often referred to as a
plasmid "mini-prep," yields fairly
clean DNA quickly and easily.
Principe of chromatography
separation of DNA on the columns
Protocol-at-a-glance
H+
DEAE diethylaminoethanol
N: N+ H
CH2CH3
CH2CH3
CH2CH3
CH2CH3
OCH2CH2 OCH2CH2
Struktura DNA
Biochemistry practical lesson 3 Isolation nd characterisation of DNA and
proteins
- 2 - DNA
3.2 UV SPECTROPHOTOMETRY OF DNA
OBJECTIVE: ANALYSIS OF DNA, DETERMINATION OF DNA CONCENTRATION
Biochemists routinely work with DNA, RNA and proteins and have devised some simple, fast
spectrophotometric assays for these molecules. The purpose of this exercise is to use the UV
absorbance of biological samples to obtain qualitative and quantitative information about
those samples. In this spectrophotometry exercise you will:
1. Produce UV absorbance spectra of DNA
2. Quantify plasmid DNA by measurement of DNA absorbance at 260 nm
1. UV Spectrophotometry of DNA, RNA, and Proteins
Brief Background Review
A. The UV Absorbance Spectra of Nucleic Acids and Proteins .
Most biological molecules do not intrinsically absorb light in the visible range, but they do absorb ultraviolet
light. Biologists take advantage of UV absorbance to quickly estimate the concentration and purity of DNA,
RNA, and proteins in a sample. In a previous laboratory, you learned how to quantify DNA using the
diphenylamine reaction. It is also possible to quantify the amount of DNA in a sample by looking at its
absorbance at a wavelength of 260nm or 280nm (in the UV region). The UV method described in this exercise is
not highly accurate but it is very widely used since it is easy, quick and little DNA is required.
Biochemistry practical lesson 3 Isolation nd characterisation of DNA and
proteins
- 3 - DNA
Proteins have two absorbance peaks in the UV region, one between
215-230 nm, where peptide bonds absorb, and another at about 280 nm
due to light absorption by aromatic amino acids (tyrosine, tryptophan
and phenylalanine). Certain of the subunits of nucleic acids (purines)
have an absorbance maximum slightly below 260 nm while others
(pyrimidines) have a maximum slightly above 260 nm. Therefore,
although it is common to say that the absorbance peak of nucleic acids
is 260 nm, in reality, the absorbance maxima of different fragments of
DNA vary somewhat depending on their subunit composition. Figure 1
shows the UV spectra for DNA, RNA, and proteins.
Protocol of spectrophotometry of DNA and proteins
1. Preparation of samples
- Dilute samples first in eppendorf tubes -see table
sample l (2-20 l pipette) water l (0,1-1 ml pipette)
1 Plasmid DNA 10 490
2 Blank - water 0 500
2. Measurement
- Prepare samples 1-2 (by pipetting) to Eppendorf tube
- Measure the absorbance of samples against deionized water
- Fill up the table
A230 A260 A280 A330 A260/
A280
A260/
A230
1 Plasmid DNA
3. Quantification of plasmid DNA concentration from absorbance at 260 nm
For calculation of pure dsDNA, a simple method is used.
If a sample containing pure double-stranded DNA has an absorbance of 1 at 260 nm in 1
cm cuvette, then it contains approximately 50 µg/mL of double-stranded DNA.
CALCULATE CONCENTRATION OF PLASMID DNA
A260=1............concentration of DNA 50 µg/mL
A260= ..........----------------......X............. concentration of DNA in cuvette
Don’t forget on dilution
Dilution = final volume (10+490l)/initial volume (10 l)
Concentration of DNA in sample = dilution * X µg/mL
Your calculation:
RNA
DNA
BSA
RNA
DNA
BSA
Biochemistry practical lesson 3 Isolation nd characterisation of DNA and
proteins
- 4 - DNA
3.3 AGAROSE ELECTROPHORESIS OF DNA
Background
Agarose gel electrophoresis is the easiest and most common way of separating and analyzing
DNA. The purpose of the gel might be to look at the DNA, to quantify it or to isolate a
particular band. The DNA is visualized in the gel by addition of ethidium bromide. This binds
strongly to DNA by intercalating between the bases and is fluorescent, meaning that it absorbs
invisible UV light and transmits the energy as visible orange light.
1. Preparing and Running Standard Agarose DNA Gels
The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively
simple and include:
 An electrophoresis chamber and power supply
 Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic.
The open ends of the trays are closed with tape while the gel is being cast, then removed prior to
electrophoresis.
 Sample combs, around which molten agarose is poured to form sample wells in the gel.
 Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
 Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the
sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or
how far the electrophoresis has proceeded.
 Ethidium bromide, a fluorescent dye used for staining nucleic acids. NOTE: Ethidium bromide is a
known mutagen and should be handled as a hazardous chemical - wear gloves while handling.
 Transilluminator (an ultraviolet lightbox), which is used to visualize ethidium bromide-stained DNA in
gels. NOTE: always wear protective eyewear when observing DNA on a transilluminator to prevent
damage to the eyes from UV light.
A) To pour a gel, 1 g of agarose powder is mixed with electrophoresis buffer (1X TAE) to the desired
concentration, then heated in a microwave oven until completely melted. Most commonly, ethidium bromide is
added to the gel (final concentration 0.5 ug/ml) at this point to facilitate visualization of DNA after
electrophoresis. After cooling the solution to about 60C, it is poured into a casting tray containing a sample
comb and allowed to solidify at room temperature or, if you are in a big hurry, in a refrigerator.
B) After the gel has solidified, the comb is removed, using care not to rip the bottom of the wells. The gel, still
in its plastic tray, is inserted horizontally into the electrophoresis chamber and just covered with buffer. Samples
containing DNA mixed with loading buffer are then pipette into the sample wells, the lid and power leads are
placed on the apparatus, and a current is applied. You can confirm that current is flowing by observing bubbles
coming off the electrodes. DNA will migrate towards the positive electrode, which is usually colored red.
C) Calculate the volumes of DNA and RNA samples needed for preparing appropriate amounts of nucleic
acids to load on gel. Complete the tables. Prepare 13 samples for electrophoresis according to the table and
load them on gel.
sample Volume of DNA l 6xLB
Plasmid DNA 5
Plasmid DNA 10
corresponding to supercoiled forms and nicked circles. The image to the right shows an EtBrstained
gel with uncut plasmid in the left lane and the same plasmid linearized at a single site
in the right lane.
After placing the gel on UV transilluminator, we can photograph it.