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SHORT COMMUNICATION 853
sec. The tubes were incubated at room temperature for The
electrophoretic profile (0.8% agarose) of these
5 min and the contents were centrifuged at 12879g, at DNA
preparations obtained using all detergents has
4 C for 10 min to get rid of the cell debris. The been shown in
Fig. 1. The presence of single bando
supernatants so obtained were transferred to other with high
intensity in all cases indicated the high
microcentrifuge tubes and the centrifugation was integrity of
DNA since there was no shearing of DNA
repeated to remove any existing residue of proteins observed on
the profile. The DNA preparationsetc. The final supernatants of two
tubes having same obtained using all detergents and Flexi gene DNA
kit
detergent, were pooled in 15 ml centrifuge tube. were used for
amplification of goat aromatase exon 2.
Absolute ethanol about 4 ml was added to pooled The results
(Fig. 2) of PCR product profile in 2%
supernatant and mixed gently for precipitation of agarose gel
indicated the presence of 352 bp band
DNA. The DNA threads were retrieved by using heat representing
exon 2 in all DNA preparations. The
sealed Pasteur pipette. The DNA on the Pasteur results suggested
that even the genomic DNA isolated
pipette was washed with 4 ml of 70% ethanol and by some of
detergents showed poor purity factor and
immediately kept in 1 ml of 10 m MTris-HCl buffer yield also,
this did not affect exon 2 amplification.
(pH 8) for proper dissolution. Then, the DNA solution This
further supports the view that these preparationsowas incubated at
60 C for 2 hr in a water bath to had DNA of high integrity.
inactivate DNases. Finally, the DNA solution was The synthetic
detergent powders consist of surfaceostored at -20 C until further
use. Similarly, the two active agents (alkyl sulphates), builders
and fillers. In
sets of WBC suspensions were processed for each addition, they
have additives like antidepositiondetergent separately used for
isolation of genomic agents, optical brighteners, bleaching agents,
foam
DNA as per above protocol.Table1 Purity and yield of DNA
isolated from goat blood using
Polymerase chain reaction (PCR) The PCR was different brands of
household detergents and Flexi gene DNA kit
performed in a total volume of 50 l of reaction [Values are
mean+ SE from 6 observations each]mixture consisting of 1X PCR
buffer [10 m MTris-
YieldHCl ( pH 9), 50 m MKCl, 1.5 m MMgCl , 0.01% Detergent brand
name Purity factor2 (A 260/A280) (g/ml of blood)gelatin], 0.15 m
Mof each dNTP. 0.2 Mof each
aAriel 1. 87 0.015 29.48 2.l2primer and 1U Taq DNA polymerase.
TheActive wheel 1. 8l 0.022 28.34 1.34 aamplification was done in a
thermocycler using cycleTide 1. 55 0.015 45.74 1.5a bconditions as
94 C for 2 min, 35 cycles of 94 C for 1Henko stain Champion 1. 8l
0.016 28.68 2.57 amin, 60 C for 1 min, 72 C for 1 minute and a
lastRin 1. 76 0.016 39.36 3.73 bcycle at 72 C for 5 min.Ezee 1. 79
0. 017 38.69 1.95 b
The data were analyzed byStatistical analysis Flexi gene DNA kit
(Control) 1. 84 0.085 44.22 3.51 bANOVA, using MS EXCEL
software.
indicate the significant difference. Critical difference for
puritya , bThe genomic DNA was isolated from goat
0.01. Critical difference for yield 6.979peripheral blood using
different detergents as
described. The purity factor (A /A ) and yield of 260 280
DNA are presented in Table 1. The results showed a
similar purity factor for DNA isolated by different
detergents except the DNA isolated by Tide which
exhibited a little impure preparation showing the
purity factor (1.550.015) below the acceptable range
(1.7-2.0). Among all the detergents used Ariel gave
the DNA of highest purity (1.870.015) even better
than that of control. However, the DNA yield with
this detergent was very poor. Similarly, Active
Wheel, Henko stain champion showed good purity
factor but lower yield of DNA. Isolation of DNAFig. l
Electrophoretic profile (0.8% agarose) of genomic DNA
using Rin and Ezee resulted in significantly higher(100 ng)
isolated from goat blood using different brands of
purity factor and yield and these were comparable detergents
(Lane 1, Ariel; Lane 2, Active wheel ; Lane 3, Tide;with that of
DNA isolated by Flexi gene DNA kit. Lane 4, Henko stain Champion ;
[Lane 5, Rin; Lane 6, Ezee].
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INDIAN J EXP BIOL, OCTOBER 2006854
enzyme, may break down and denature the proteins.
Addition of 5 MNaCI precipitates the denatured
proteins due to salting out. In addition, alkyl sulphates4which
are anionic surfactants may separate
negatively charged DNA molecules from other
molecules. The differences in the purity and yield ofDNA between
different detergents may be due to
difference in the proportion of above compounds.
Though it seems Rin and Ezee are best as far as purity
and yield are concerned, there is no difference in
downstream processing (PCR, the heart of molecular
biology) among all detergents, suggesting that all the
detergents used in present study can be recommended
for DNA isolation and downstream applications such
as PCR to small laboratories having budget
constraints. This method is highly economic and
efficient, because the use of proteinase K, phenol,Fig. 2
Electrophoretic profile (2% agarose) of amplified goat
guanidinium hydrochloride which is commonly usedaromatase (exon
2) genomic DNA: The PCR was performed as
in standard DNA isolation protocols, are no moreper protocol
described [Lane 1, 100 bp DNA ladder; Lane 2,required.3,4,5,6,7 and
8 represent the PCR products after amplification of
DNA isolated by Flexi gene DNA kit, Ariel, Active Wheel,
Tide,
Henko stain Champion, Rin and Ezee, respectively].References
1 Bahl A & Pfenninger M, A rapid method of DNA
isolationregulators, organic sequestering agents, enzymes using
laundry detergent, Nucleic Acids Res , 24 (1996) 1587.(subtilisin,
cellulase), perfumes, and substances to 2 Drabek J & Petrek M,
A sugar laundry detergent and saltregulate density and assure
crispiness of the clothes. method for extraction of
deoxyribonucleic acid from blood,
Biomed Papers , 146 (2002) 37.During DNA isolation, by adding
detergent solution3 How green your detergent? Web:
http://www.consumer-
the WBC will be lysed due to hyper osmosis andvoice.org/
vigorous shaking. The detergent organic sequestering 4 MSDS #
FH/H/2002/GLGL-59YQJN, Tide granular Laundry4agents and enzymes
such as subtilisin , a proteolytic Detergent MSDS.Doc.P&G.s
