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A wide-range, low-cost 150 bp ladder for sizing DNA fragments between 150 and 4500 bp

A wide-range, low-cost 150 bp ladder for sizing DNA fragments between 150 and 4500 bp

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Published by clfront
Zeev Barvish
Claytus Davis
Inna Gitelman
Department of Virology and
Developmental Genetics,
Faculty of Health Sciences,
Ben Gurion University
of the Negev,
Beer Sheva, Israel
Received July 15, 2006
Revised September 10, 2006
Accepted September 20, 2006
Short Communication
A wide-range, low-cost 150 bp ladder
for sizing DNA fragments between 150
and 4500 bp
Agarose gel electrophoresis, a very routine procedure, requires molecular weight standards;
these are usually manufactured from plasmid or viral DNA fragments, or more recently,
from PCR products of defined sizes. We describe here the preparation of a molecular
weight standard from a completely different DNA source – the uniquely organized genome
of the beetle Tenebrio molitor. The standard can be used to accurately size DNAs between 150
and 4500 bp, a useful range of sizes for many agarose gel electrophoresis applications,
including separation of PCR products and plasmid cloning targets. In addition, it is easy to
prepare, inexpensive, and rivals the best of the commercial ladders.
Keywords:
Molecular weight standard / Nucleic acid / Satellite DNA / Size marker
DOI 10.1002/elps.200600438 Electrophoresis 2007, 28, 900–902
Zeev Barvish
Claytus Davis
Inna Gitelman
Department of Virology and
Developmental Genetics,
Faculty of Health Sciences,
Ben Gurion University
of the Negev,
Beer Sheva, Israel
Received July 15, 2006
Revised September 10, 2006
Accepted September 20, 2006
Short Communication
A wide-range, low-cost 150 bp ladder
for sizing DNA fragments between 150
and 4500 bp
Agarose gel electrophoresis, a very routine procedure, requires molecular weight standards;
these are usually manufactured from plasmid or viral DNA fragments, or more recently,
from PCR products of defined sizes. We describe here the preparation of a molecular
weight standard from a completely different DNA source – the uniquely organized genome
of the beetle Tenebrio molitor. The standard can be used to accurately size DNAs between 150
and 4500 bp, a useful range of sizes for many agarose gel electrophoresis applications,
including separation of PCR products and plasmid cloning targets. In addition, it is easy to
prepare, inexpensive, and rivals the best of the commercial ladders.
Keywords:
Molecular weight standard / Nucleic acid / Satellite DNA / Size marker
DOI 10.1002/elps.200600438 Electrophoresis 2007, 28, 900–902

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Published by: clfront on Feb 11, 2009
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Zeev BarvishClaytus DavisInna Gitelman
Department of Virology andDevelopmental Genetics,Faculty of Health Sciences,Ben Gurion Universityof the Negev,Beer Sheva, IsraelReceived July 15, 2006Revised September 10, 2006Accepted September 20, 2006
Short Communication
Awide-range, low-cost 150 bp ladderfor sizing DNA fragments between 150and 4500 bp
Agarose gel electrophoresis,a very routine procedure,requires molecular weight standards;these are usually manufactured from plasmid or viral DNA fragments, or more recently,from PCR products of defined sizes. We describe here the preparation of a molecularweight standard from a completely different DNA source – the uniquely organized genomeofthe beetle
Tenebrio molitor 
. The standard can beused to accurately sizeDNAs between150and 4500 bp, a useful range of sizes for many agarose gel electrophoresis applications,including separation of PCR products and plasmid cloning targets. In addition, it is easy toprepare, inexpensive, and rivals the best of the commercial ladders.
Keywords:
Molecular weight standard / Nucleic acid / Satellite DNA / Size markerDOI 10.1002/elps.200600438
900
Electrophoresis 
2007,
28,
900–902
As much as 40–60% of the genome of the beetle
Tenebriomolitor 
consists of a highly homogeneous centromeric satel-lite DNA composed of close to ten million copies of a tan-demly repeated 142 bp fragments. The consensus monomersequence contains one
Eco
RI restriction endonuclease site[1–3]. It may be possible to take advantage of this abundantand uniform satellite to produce a good nucleic acid sizestandard, since a partial restriction enzyme digestion of tan-demly repeated sequences produces a ladder of bands with astep distance equal to the length of the repeat,
e.g.
[4, 5]. Cer-tainly, partial
Eco
RI digestion produces ladders (Fig. 1) thatcould potentially be used as molecular weight markers [1, 6].Yet, beforeasserting that a ladder is both useful and up to thestandards of the many commercial markers, two issues needresolution. First, the migration of repetitive sequences dur-ing DNA gel electrophoresis must be calibrated and second,it is necessary to verify that the nonsatellite portion of thegenomic DNA does not obscure the ladder.The issue of calibration arises because some satelliteDNAs, including that of 
T. molitor 
[7], exhibit anomalousmigration velocitiesduring gel electrophoresis[8–10]. This isprobably due to DNA curvature–the formation of a tertiarystructure that can retard DNA motility, which is especiallycommon in tandemly repeated sequences.To evaluate and calibrate the mobility of the bands in the
T. molitor 
ladder, we prepared a partial digest and electro-phoresed it on a standard 1% agarose slab gel beside fourdifferent commercially available ladders (Fig. 2). We thenproduced standard semi-log plots of log
10
size againstmigration distance. The higher resolution plot of the 700–3000 bp range size (Fig. 3) showed that migration of the
T.molitor 
DNA fragments (Fig. 3, open circles) was consistentlyretarded compared to those of the other ladders. However,the curves of 
T. molitor 
DNA and of the commercial markerswere very similar (Fig. 3, inset) and very nearly parallel.Nonlinear regression was used to model the migration ofthecommercial markers in order to determine the neededadjustment to the
Tenebrio
ladder. According to the regres-sion analysis, the partially cleaved
T. molitor 
satellite DNAruns as a 150 bp DNA ladder, with rung sizes of 150, 300,450 bp,
etc.
that is very nearly superimposed on the com-mercial ladders (Fig. 3, closed circles). It must be noted thatthere are slight differences in the curves of all the ladders,which means that for the
Tenebrio
ladder (closed circles) at3000 bp, an underestimation of size by 50 or 80 bp wouldoccur if the Hyperladder 1 or the O’RangeRuler, respectively,is regarded as absolutely accurate.The secondissue that had to be addressedwas the degreeto which the nonsatellite portion of the
Tenebrio
DNA inter-fered with the marker. The ladder is a result of partial diges-tion of the unusually abundant tandemly repeated satelliteDNAthat constitutesabouthalf ofthe
T. molitor 
genome.Thequestion arose whether the other, nonsatellite, half of thegenome, where the
Eco
RI sites are distributed randomly,would produce a smear that obscured the ladder. The smear
Correspondence:
Dr. Inna Gitelman, Department of Virology andDevelopmental Genetics, Faculty of Health Sciences, Ben GurionUniversity of the Negev, Beer Sheva, 84105, Israel
E-mail:
gitelman@bgu.ac.il
©
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.electrophoresis-journal.com
 
Electrophoresis 
2007,
28,
900–902
Nucleic Acids
901
Figure 1.
Partial and complete digest of
T. molitor 
genomic DNAwith
Eco 
RI.
T. molitor 
genomic DNA (35
m
g) was digested with30 U of
Eco 
RI restriction endonuclease at 37
7
C. Aliquots (10
m
L)wereremovedatdifferenttimes(below).Forthecompletedigest,another 50 U of the enzyme was added and the reaction con-tinued for another 30 min. Samples were electrophoresed on a1% agarose gel and visualized with ethidium bromide staining.Sizes aregivenin kbp. Lane1 –phage lambda DNAdigested with
Hin
dIII, lanes 2–11 – reaction samples from times
= 0, 1.5, 5, 10,15, 20, 30, 35, and 40 min respectively, and lane 12 – completedigest.
was indeed visible(Fig. 2, lane 1), but it started to obscurethebands only at sizes larger than 4500 bp, leaving almost theentire resolvable ladder unaffected.In summary, the 150 bp ladder can be used to accuratelyestimate DNA fragment sizes between 150 and 4500 bp, arange that covers most PCR products and plasmid cloningtargets. The ladder matches the best of the commercialmarkers, and at molecular weights above 1500 bp, it clearlypermits more accurate immediate size estimates than thecommercial ones tested, with the exception, perhaps, of theO’RangeRuler.Making the ladder:
T. molitor 
larvae, commonly calledmealworms, are used as fish bait and food for lizards andamphibians and are available in most pet stores. Extraction
Figure 2.
Partial
Eco 
RI digest of
T. molitor 
genomic DNA gen-erates a ladder for accurate sizing of nucleic acids in the range of150 to 4500 bp. The
T. molitor 
ladder was prepared as described.DNAs were electrophoresed on a 1% agarose gel and visualizedwith Syber Gold (Molecular Probes) staining and UV illumina-tion. Lane 1:
T. molitor 
ladder; lane 2: 2
m
L Hyperladder I (BioLineBIO-33025); lane 3: 1
m
L 100 bp ladder (Invitrogen 15628-019);lane 4: 1
m
L 100 bp ladder (NEB N3231S); lane 5: 5
m
L O’Ran-geRuler 100 bp DNA ladder (MBI/Fermentas, SM0623).
of genomic DNA followed the standard technique [11].Briefly, final instar larvae, approximately 2–3 cm long, werefrozen in liquid nitrogen or in dry ice, and pulverized withmortar and pestle. Following a standard proteinaseK/SDSdigest, three phenol/chloroform (1:1) extractions were doneand the DNA precipitated with ethanol. Two dozen 2–3 cmlong larvae yield 2 mg of purified DNA. The optimal condi-tions for obtaining a good ladder were determined empiri-cally for each batch of DNA. We used between 0.05 and 1 Uof 
Eco
RI
per 
1
m
g of DNA at 37
7
C and assessed cutting attimes ranging between 1 min and 2 h, with aliquots of ap-proximately 1
m
g taken at each time interval for testing on agel. Typically, a 1 h digest at these conditions gave the bestladder. To further improve the ladder, we mixed four parts of partial DNA digest (Fig. 1, lane 5) with one part of completedigest, which produces mostly the monomer band (Fig. 1,lane 12). While in the most useful partial digest (Fig. 1, lane5) the smallest band stains weakly, addition of extra mono-mer shifts itsstoichiometryand producesa ladder with a verybright band at its leading end (Fig. 2, lane 1). Presence ofthisunusually bright band validates the presence of the entire
©
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.electrophoresis-journal.com

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