Dna quantification

this section helps students how to quanify the isolated DNA by spectrophotometer. specially life life science fields such as biotechnology, biology, and medical laboratory

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
ReliablemeasurementofDNAconcentrationisimportant
formanyapplications
DNAquantityandqualitycanbeassessedusingseveral
differentmethodsinclude:
AbsorbancebyspectrophotometerorNanophotometer.
Agarosegelelectrophoresis.
Absorbance: is the most common easies to
determine DNA yield and purity.
Tadele T, April 2010 E.C

4. QualityofDNAusingspectrophotometer
•An instrument employed to measure the amount of
light that a sample absorbs.
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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:Nucleicacidsabsorblightat260nm,theA260readingshould
bebetween0.1–1.0.Thespectrophotometerismostaccuratewhen
measurementsareintherangeof0.1–1.0.
However,DNAisnottheonlymoleculethatcanabsorbUV-
lightat260nm.
SinceRNAalsohasagreatabsorbanceat260nmwill
contributetothetotalmeasurementat260nm
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7. Theratiooftheabsorbanceat260nm/280nmisa
measureofthepurityofaDNA;itshouldbebetween
1.7and2.0.
If < 1.7, the nucleic acid preparation may be contaminated with
protein. Use protinaseKto remove protein.
If > 2.0 indicates RNA contamination. RNase should be used to
remove the contaminating RNA.
DNAPurity(A260/A280)=(A260reading–A320reading)
/(A280reading–A320reading)
7Tadele T, April 2010 E.C

8. Theratiooftheabsorbanceat260nm/320nmisameasure
ofthepurityofaDNAsamplefromorganicsand/orsalts;
itshouldbeabout2.0.
LowA260/A320ratioindicatescontaminationbyorganics
and/orsalts.
Theabsorbancereadingindicateshowmuchthesampleispure.
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9. QuantificationofDNAbyspectrophotometry.
UsingTEbufferasthediluent,
MakeanappropriatedilutionofyourDNAdependingon
thesizeofthecuvettesavailable(e.g.for1mlcuvettes,
dilute10microliterDNAsolutionin990microlitersof
TE).
DeterminetheabsorbanceofDNAat260nmusingTEasthe
referencesolution(i.e.asablank).
9Tadele T, April 2010 E.C

10. Usingaconversionfactor:
◦oneODat260nmisequivalentto
Multiplytheabsorbancereadingby
theconversionfactorand
thedilutionfactortofindtheconcentrationofnucleic
acid.
PureDNAConcentration(microg/ml)=
(A260reading–A320reading)xdilutionfactorx50microg/ml
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11. Total yieldis obtained by multiplying the
DNA concentration by the final total purified
sample volume.
DNA Yield (microgram/ml)= DNA
Concentration x Total Sample Volume
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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 50µL 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 50µL 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?
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Don’tneeddilution
Thevolumerequiredformeasurement3-5
microliters
Theconcentrationgiveninnanogram
\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
Runningyoursamplethroughanagarosegelisa
commonmethodforexaminingtheextentofDNA
degradation.
Smearingindicates
DNAdegradationor
ToomuchDNAloaded.
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GoodqualityDNAshould
migrateasahighmolecular
weightband,withlittleorno
evidenceofsmearing.

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Thank you
Tadele T, April 2010 E.C