familarize students how to quantify and determine DNA

1. DNA quantification & purity
determination
1Tadele T, April 2010 E.C

2. University of Gondar
Institute of Biotechnology
Techniques in Biotechnology (Biot.602)
Lecture 4
DNA quantification & purity determination

3. 3
 Reliable measurement of DNA concentration is important
for many applications
 DNA quantity and quality can be assessed using several
different methods include:
Absorbance by spectrophotometer or Nanophotometer.
Agarose gel electrophoresis .
 Absorbance: is the most common easies to
determine DNA yield and purity.
Tadele T, April 2010 E.C

4. Quality of DNA using spectrophotometer
• An instrument employed to measure the amount of
light that a sample absorbs.
4Tadele T, April 2010 E.C

5. 5
 The rings of the bases (A, C, G, T, U)
are made up of alternating single
and double bonds.
 Such ring structures absorb in
the U.V.
 Each of the four nucleotide
bases has a slightly different
absorption spectrum, and
 The spectrum of DNA is
the average of them.
Tadele T, April 2010 E.C

6. ◦ DNA UV absorbance at 260nm.
◦ protein >> at 280nm.
◦ Carbohydrate >> at 230nm.
◦ Any insoluble light-scattering components……. absorbance at
320 nm.
Note: Nucleic acids absorb light at 260 nm ,the A260 reading should
be between 0.1–1.0. The spectrophotometer is most accurate when
measurements are in the range of 0.1–1.0.
 However, DNA is not the only molecule that can absorb UV-
light at 260nm.
Since RNA also has a great absorbance at 260nm will
contribute to the total measurement at 260nm
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7.  The ratio of the absorbance at 260 nm/280 nm is a
measure of the purity of a DNA; it should be between
1.7 and 2.0.
 If < 1.7, the nucleic acid preparation may be contaminated with
protein. Use protinase K to remove protein.
 If > 2.0 indicates RNA contamination. RNase should be used to
remove the contaminating RNA.
 DNA Purity (A260/A280) = (A260 reading – A320 reading)
/(A280 reading – A320 reading)
7Tadele T, April 2010 E.C

8.  The ratio of the absorbance at 260 nm/320 nm is a measure
of the purity of a DNA sample from organics and/or salts;
it should be about 2.0.
 Low A260/A320 ratio indicates contamination by organics
and/or salts.
 The absorbance reading indicates how much the sample is pure.
8Tadele T, April 2010 E.C

9. Quantification of DNA by spectrophotometry.
 Using TE buffer as the diluent,
 Make an appropriate dilution of your DNA depending on
the size of the cuvettes available (e.g. for 1ml cuvettes,
dilute 10 microliter DNA solution in 990 micro liters of
TE).
 Determine the absorbance of DNA at 260 nm using TE as the
reference solution (i.e. as a blank).
9Tadele T, April 2010 E.C

10.  Using a conversion factor :
◦ one OD at 260 nm is equivalent to
 Multiply the absorbance reading by
 the conversion factor and
 the dilution factor to find the concentration of nucleic
acid.
 Pure DNA Concentration (microg/ml) =
(A260 reading – A320 reading) x dilution factor x 50microg/ml
10Tadele T, April 2010 E.C

11.  Total yield is obtained by multiplying the
DNA concentration by the final total purified
sample volume.
 DNA Yield (microgram/ml) = DNA
Concentration x Total Sample Volume
11Tadele T, April 2010 E.C

12.  Problem. From a small culture, you have purified the DNA of a
recombinant plasmid. Then you have resuspended the DNA in a
volume of 50 µL TE. You dilute 20 µL of the purified DNA sample
into a total volume of 1000 µL distilled water. You measure the
absorbance of this diluted sample at 260 nm and 280 nm and obtain
the following readings.
A260 --- 0 . 5 5 0
A280 - 0 . 3 2 4
a) What is the DNA concentration of the 50 µL plasmid prep?
b) How much total DNA was purified by the plasmid prep
procedure?
c) What is the A260/280 ratio of the purified DNA?
12Tadele T, April 2010 E.C

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 Don’t need dilution
 The volume required for measurement 3-5
microliters
 The concentration given in nanogram
microliters.
Tadele T, April 2010 E.C

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 Quality of DNA extracted is assessed using
the following simple protocol:
 Mix 3µL of DNA with 12µL of loading
Dye
 Load this mixture into a 1% agarose gel
 Stain with ethidium bromide
 Electrophorese at 70–80 volts, 45–90
minutes.
Tadele T, April 2010 E.C

15. Checking for Degradation DNA
 Running your sample through an agarose gel is a
common method for examining the extent of DNA
degradation.
 Smearing indicates
 DNA degradation or
 Too much DNA loaded.
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Good quality DNA should
migrate as a high molecular
weight band, with little or no
evidence of smearing.

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Thank you
Tadele T, April 2010 E.C