Purified DNA Fragments Research Articles

Statistical design approach enables optimised mechanical lysis for enhanced long-read soil metagenomics

Metagenomic analysis has enabled insights into soil community structure and dynamics. Long-read sequencing for metagenomics can enhance microbial ecology by improving taxonomic classification, genome assembly, and functional annotation. However, protocols for purifying high-molecular weight DNA from soil are not yet optimised. We used a statistical design of experiments approach to enhance mechanical lysis of soil samples, increasing the length of purified DNA fragments. Low energy input into mechanical lysis improved DNA integrity, resulting in longer sequenced reads. Our optimized settings of 4 m s−1 for 10 s increased fragment length by 70% compared to the manufacturer’s recommendations. Longer reads from low intensity lysis produced longer contiguous sequences after assembly, potentially improving a range of down-stream analyses. Importantly, there was minimal bias exhibited in the microbial community composition due to lysis efficiency variations. We therefore propose a framework for improving the fragment lengths of DNA purified from diverse soil types, improving soil science research with long-read sequencing.

Molecular Identification of Dominant Microbes in Kola nut (Cola nitida)

Kola nut is an important dehiscent fruit but its supply fails to meet the demand due to limitations such as inadequate post-harvest practices leading to spoilage during transportation and storage. Identification of microbial contaminants is a necessary step in reducing the loss. Hence, this work aimed at characterizing the genome of dominant fungus and bacteria associated with kola nut (Cola nitida). Samples of kola nuts were collected from Oje and Oja-oba markets in Ibadan, Nigeria. Isolates were extracted from the lesion region of the samples using the standard method. Macro-morphological and micro-morphological observations of the pure culture of the isolates were conducted. After genomic DNA extraction of fungal and bacterial isolates, Polymerase Chain Reaction (PCR) was carried out using Internal Transcribed Sequence (ITS 1 and ITS 4) and 27F primers for fungal and bacterial genome respectively. Sanger sequencing of the purified DNA fragments was conducted utilizing the Nimagen, BrilliantDye™ Terminator Cycle Sequencing Kit V3.1, BRD3-100/1000. Molecular Evolutionary Genetics Analysis (MEGA version 6) was used for phylogenetic analysis. The result revealed that one fungus and two bacteria were associated with kola nuts. The fungal isolate showed 100% identity with Aspergillus wentii from the National Center for Biotechnology Information data base. The fungus showed a good relationship with A. dimorphicus and A. europaeus. The two bacterial isolates were also identified through the phylogenic analysis as Bacillus cereus and B. subtilis. B. cereus is closely related to B. thuringiensis. This research provides background information on the Kola nut microbial contaminant. Keywords: Bacteria, Contaminant, Fungus, Identification, Primers, Sequencing

Cytoplasmic Microinjection of <i>piggyBac</i> Transposase mRNA and Transposon Vectors for Efficient <i>In Vitro</i> Production of Transgenic Porcine Parthenotes

The efficient production of transgenic (Tg) piglets has remained a challenge in the field of domestic animal studies. Unlike mice, the pronuclei of pig zygotes cannot be easily studied because of the abundance of lipid droplets. Therefore, the zygotes must be briefly centrifuged before pronuclear injection (PNI) to move the lipid droplets to the periphery of the zygote for PNI-mediated production of Tg piglets. However, this procedure is temporal because lipid droplets return to the original space during PNI, hampering the consecutive PNI. Cytoplasmic injection (CPI) of nucleic acids is comparatively simple than PNI because CPI does not require such pre-centrifugation. Unfortunately, CPI using purified DNA fragments is inadequate for creating Tg piglets because it is challenging to integrate nucleic acids into the host genome. <em>PiggyBac</em> (PB), one of the transposons, is a valuable tool enabling efficient chromosomal integration of a transgene. The PB-mediated gene transfer requires two components, namely, transposase and transposons harboring the gene of interest (GOI) flanked by PB acceptor sites. We speculate that the CPI of transposase mRNA and transposons could accelerate the chromosomal integration of the GOI in pig zygotes. To prove this hypothesis, we performed CPI using transposase mRNA (super PB transposase mRNA) + transposon DNA carrying the enhanced green fluorescent protein (<em>EGFP</em>) cDNA (referred to as “pT-EGFP”), transposase expression plasmid DNA (referred to as “pTrans”) + pT-EGFP, pT-EGFP alone, or non-transposon EGFP expression plasmid DNA using porcine parthenotes. Consequently, 50% (2/4 tested) of green-fluorescent embryos exhibited chromosomal integration of GOI. In contrast, green-fluorescent embryos derived from CPI with pTrans + pT-EGFP or pT-EGFP alone did not show chromosomal integration. We used mRNA for super PB transposase, which is an engineered hyperactive version of the wild-type PB transposase. To conclude, the PB system, based on the CPI of super PB transposase mRNA + transposon DNA, could be useful for producing Tg porcine parthenotes.

Random Priming: Labeling of Purified DNA Fragments by Extension of Random Oligonucleotides.

In this protocol, oligonucleotides that are heterogeneous in sequence serve as primers for the initiation of DNA synthesis on single-stranded templates. Labeled dNTPs ([α-32P]dNTPs or biotin-, DIG-, or fluorescein-labeled dUTPs) are incorporated into the new DNA by the Klenow fragment of Escherichia coli DNA polymerase I (Pol I). The method can be adapted to radiolabel DNA in slices cut from gels cast with low-melting-temperature agarose.

Analysis of DNA by Agarose Gel Electrophoresis.

Electrophoresis through agarose or polyacrylamide gels is used to separate, analyze, identify, and purify DNA fragments. The technique is simple, rapid to perform, and capable of resolving fragments of DNA that cannot be separated adequately by other procedures, such as density gradient centrifugation. The location of bands of DNA within the gel can be determined directly by staining with low concentrations of fluorescent intercalating dyes, such as ethidium bromide or SYBR Gold; bands containing as little as 20 pg of double-stranded DNA can then be detected by direct examination of the gel in ultraviolet (UV) light. If necessary, these bands of DNA can be recovered from the gel.

Antenna size reduction and altered division cycles in self-cloned, marker-free genetically modified strains of Haematococcus pluvialis

The microalga Haematococcus pluvialis (H. pluvialis) is widely used for the commercial production of the red high value pigment astaxanthin. The major limitations of this species for supplying cheap natural astaxanthin in large scale cultivation are its slow growth rate and sensitivity to contaminants. We have applied recently developed advanced tools to genetically engineer H. pluvialis, by inserting two genes, the low CO2 inducible gene lciA (predicted to be a bicarbonate transporter) and mutated nucleic acid binding protein I, nabI* (a constitutively active translational repressor of the LHCII proteins), which were previously reported to enhance biomass productivity when overexpressed in various microalgae. Both genes were successfully integrated into the H. pluvialis genome, either separately or together, using the endogenous norflurazon resistant mutated gene of phytoene desaturase as a selection marker. Successful incorporation and expression of both genes using a linear purified DNA fragment, containing exclusively rearranged lciA, mutated pds and nabI* genes of H. pluvialis, represents to our knowledge the first functional self-cloning approach in microalgae, a breakthrough that can facilitate the widespread use of genetically altered microalgae. Initial detailed characterization of the growth physiology of two transformed strains revealed important information as to the impact of those two genes. Overexpression of the lciA gene alone resulted in alterations in growth physiology and division cycle, but with little impact on biomass productivity. A transformed strain expressing both lciA and mutated nabI* displayed a significant reduction in chlorophyll content, an altered division cycle and possibly enhanced biomass and carotenoid productivity under certain conditions. However, further work and additional progress exploring an array of optimizing options will be required towards creation of modified strains of truly advanced biotechnological potential for commercial applications.

Identification and Molecular Cloning of Heat Shock Protein-70 (HSP-70) Gene of Trypanosoma evansi Isolated from Camel

Present study was carried out to isolate the Heat Shock Protein-70 gene of Trypanosoma evansi using PCR. The desired amplicons of Heat Shock Protein-70 gene from genomic DNA of T. evansi were successfully amplified by PCR using gene specific primers at annealing temperature of 54°C. Amplified PCR product was identified on the basis of its size in agarose gel electrophoresis as 1956 bp. For cloning the purified DNA fragment was ligated to the pGEM-T Easy vector and ligated mixture was transformed into Escherichia coli JM109 strains. The cells containing recombinant plasmid were identified on the basis of white/blue colony selection on LB agar containing X-Gal, IPTG and ampicillin. Screening of recombinant was done by Restriction Enzyme digestion of plasmid DNAs using EcoRI and confirmed on the basis of gene size, i. e. 1956 bp for Heat Shock Protein-70 gene. Colony PCR was done for quick screening of plasmid inserts directly from E. coli colonies in the presence of insert specific primers.

Identification and molecular cloning of cysteine protease gene of Trypanosoma Evansi Isolated from Camel

A molecular study was carried out to isolate cysteine protease gene of Trypanosoma evansi using PCR. The desired amplicons of cysteine protease gene from the genomic DNA of T. evansi were successfully amplified by PCR using gene specific primers at annealing temperature of 55°C. Amplified PCR product was identified on the basis of its size in agarose gel electrophoresis as 1533 bp. For cloning the purified DNA fragment was ligated to the pGEM-T Easy vector and the ligated mixture was transformed into Escherichia coli JM109 strains. The cells containing recombinant plasmid were identified on the basis of white/blue colony selection on LB agar containing X-Gal, IPTG and ampicillin. Screening of recombinants was done by restriction enzyme digestion of plasmid DNA using EcoRI and confirmed on the basis of gene size, i. e. 1533 bp for cysteine protease gene. Colony PCR was done for quick screening of plasmid inserts directly from E. coli colonies in the presence of insert specific primers.

Optimation of chromatin immunoprecipitation technology in prostate cancer cells

Objective To optimize the key conditions of chromatin immunoprecipitation (ChIP) technology so as to improve the efficiency of the experiments. Methods Two prostate cancer cell lines (PC-3, and DU145) were chosen as the experimental materials, and some respects of volume of cracking liquid, ultrasonic power, pulse and working time were adjusted. Following conditions were set up: 25% or 30% maximum power, 35 s or 50 s interval, and 3 s or 5 s working time. The purified DNA fragments were enriched, and their concentrations and purity were detected. Polymerase chain reaction (PCR) and agarose electrophoresis were performed to check the results. Results The appropriate ChIP experimental conditions in prostate cancer cells were as follows: (1) working time of 3 s every 38 s or 5 s every 55 s with a 5 mm microtip at a power setting of 30% of the maximum power, in total time from 45 s to 75 s; (2) If the concentration of purified DNA could be in more than 10 ng/μl or purity value (A260/A280) in 1.45 above, we could get a better result. Conclusion In prostate cancer cells (PC-3, and DU145), when we selected the appropriate ultrasonic frequency, the general ultrasonic working time was about 60 s and the right DNA fragments could be obtained. Fixing volume of elution liquid, it was worth centrifugal twice than one to obtain a higher DNA purity value. What’s more, the higher purity of DNA, the more obvious of results we could arrive at. Key words: Prostate cancer; Chromatin immunoprecipitation

Capture Hybridization Analysis of DNA Targets.

There are numerous recent cases where chromatin modifying complexes associate with long noncoding RNA (lncRNA), stoking interest in lncRNA genomic localization and associated proteins. Capture Hybridization Analysis of RNA Targets (CHART) uses complementary oligonucleotides to purify an RNA with its associated genomic DNA or proteins from formaldehyde cross-linked chromatin. Deep sequencing of the purified DNA fragments gives a comprehensive profile of the potential lncRNA biological targets in vivo. The combined identification of the genomic localization of RNA and its protein partners can directly inform hypotheses about RNA function, including recruitment of chromatin modifying complexes. Here, we provide a detailed protocol on how to design antisense capture oligos and perform CHART in tissue culture cells.

RNAi-mediated knockdown of inhibin α subunit increased apoptosis in granulosa cells and decreased fertility in mice

Inhibin α (INHα), a member of TGFβ superfamily, is an important modulator of reproductive function that plays a vital role in follicular changes, cell differentiation, oocyte development, and ultimately in mammalian reproduction. However, the role of inhibin α in female fertility and ovarian function remains largely unknown. To define its role in reproduction, transgenic mice of RNAi-INHα that knock down the INHα expression by shRNAi were used. Inhibin α subunit gene was knocked down successfully at both transcriptional and translational levels by RNAi PiggyBac transposon (Pbi) mediated recombinant pshRNA vectors and purified DNA fragments were microinjected into mouse zygotes. Results showed that transgenic female mice were sub-fertile and exhibited 35.28% reduction in litter size in F1 generation relative to wild type. The decreased litter size associated with the reduction in the number of oocytes ovulated after puberty. Serum INHα level was significantly decreased in both 3 and 6 weeks; whereas, FSH was significantly increased in 3 weeks but not in 6 weeks. Furthermore, suppression of INHα expression significantly promoted apoptosis by up-regulating Caspase-3, bcl2, INHβB and GDF9 and down regulated Kitl and TGFβRIII genes both at transcriptional and translational levels. Moreover, it also dramatically reduced the progression of G1 phase of cell cycle and the number of cells in S phase as determined by flow cytometer. These results indicate that suppression of INHα expression in RNAi-transgenic mice leads to disruption of normal ovarian regulatory mechanism and causes reproductive deficiencies by promoting cellular apoptosis, arresting cellular progression and altering hormonal signaling.

A Role for Peroxisome Proliferator-activated Receptor γ Coactivator 1 (PGC-1) in the Regulation of Cardiac Mitochondrial Phospholipid Biosynthesis

The energy demands of the adult mammalian heart are met largely by ATP generated via oxidation of fatty acids in a high capacity mitochondrial system. Peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1)-α and -β serve as inducible transcriptional coregulators of genes involved in mitochondrial biogenesis and metabolism. Whether PGC-1 plays a role in the regulation of mitochondrial structure is unknown. In this study, mice with combined deficiency of PGC-1α and PGC-1β (PGC-1αβ(-/-)) in adult heart were analyzed. PGC-1αβ(-/-) hearts exhibited a distinctive mitochondrial cristae-stacking abnormality suggestive of a phospholipid abnormality as has been described in humans with genetic defects in cardiolipin (CL) synthesis (Barth syndrome). A subset of molecular species, containing n-3 polyunsaturated species in the CL, phosphatidylcholine, and phosphatidylethanolamine profiles, was reduced in PGC-1αβ-deficient hearts. Gene expression profiling of PGC-1αβ(-/-) hearts revealed reduced expression of the gene encoding CDP-diacylglycerol synthase 1 (Cds1), an enzyme that catalyzes the proximal step in CL biosynthesis. Cds1 gene promoter-reporter cotransfection experiments and chromatin immunoprecipitation studies demonstrated that PGC-1α coregulates estrogen-related receptors to activate the transcription of the Cds1 gene. We conclude that the PGC-1/estrogen-related receptor axis coordinately regulates metabolic and membrane structural programs relevant to the maintenance of high capacity mitochondrial function in heart.

Genetic Diversity of Selected Commercial Freshwater Fishes Based On Phospholipase C Zeta (Plc) Expression

Egg activation is important to help release the egg from meiotic arrest and blockpolyspermy. It is linked with an increase in intracellular egg calcium ions (Ca 2+ ) in almost all species studied and current studies imply that the mammalian sperm factor involved is a sperm-specific phospholipase C zeta, PLC ζ. Here, we report the identification of PLCζ in the testis and egg of LampamJawa. Our findings provide the evidence that PLCζ is present in the species of male and female LampamJawa (Barbonymusgonionotus). For this study, six types of commercial freshwater fish were selected i.e. Red Tilapia (Oreochromis sp. Red Tilapia), Black Tilapia (Oreochromismossambicus), Catfish or Keli (Ictaluruspunctatus), Silver Catfish or Patin (Pangasiuspangasius), Snakehead Fish or Haruan (Channastriatus) and Silver Barb or LampamJawa (Barbonymusgonionotus). The objectives of this study were to isolate the mRNA from the gonads of freshwater fishes, to identify and amplify the phospholipase C zeta (PLCζ) gene fragments, to sequence the purified DNA fragments and to compare the PLCζ sequence to other PLCζ sequence available in NCBI database. For the study of PLCζ expression, male and female LampamJawa showed bands at around 420bp in agarose gel electrophoresis suggesting the presence of PLCζ gene while no significant bands were found in other types of fishes used in this study.

Retina-specific gene excision by targeted expression of Cre recombinase

The use of Cre recombinase for conditional targeting permits the controlled removal or activation of genes in specific tissues and at specific times of development. The Rho–Cre mice provide an improved tool for studying gene ablation in rod photoreceptor cells. To establish a robust expression of Rho–Cre transgenic mice that would be useful for the study of various protein functions in photoreceptor cells, a total 11,987kb fragment (pNCHS4 Rho–NLS–cre) containing human rhodopsin promoter was cloned. The Rho–Cre plasmid was digested with EcoR1 and I Ceu-1, and the 9.316kb fragment containing the hRho promoter and Cre recombinase gel was purified. To generate transgenic mice, the purified DNA fragment was injected into fertilized oocytes according to standard protocols. ROSA26R reported the steady expression of Rho–Cre especially in photoreceptor cells, allowing further excising proteins in rod photoreceptors across the retina. This Rho–Cre transgenic line should thus prove useful as a general deletor line for genetic analysis of diverse aspects of retinopathy.

An Extra Peptide within the Catalytic Module of a β-Agarase Affects the Agarose Degradation Pattern

Agarase hydrolyzes agarose into a series of oligosaccharides with repeating disaccharide units. The glycoside hydrolase (GH) module of agarase is known to be responsible for its catalytic activity. However, variations in the composition of the GH module and its effects on enzymatic functions have been minimally elucidated. The agaG4 gene, cloned from the genome of the agarolytic Flammeovirga strain MY04, encodes a 503-amino acid protein, AgaG4. Compared with elucidated agarases, AgaG4 contains an extra peptide (Asn(246)-Gly(302)) within its GH module. Heterologously expressed AgaG4 (recombinant AgaG4; rAgaG4) was determined to be an endo-type β-agarase. The protein degraded agarose into neoagarotetraose and neoagarohexaose at a final molar ratio of 1.5:1. Neoagarooctaose was the smallest substrate for rAgaG4, whereas neoagarotetraose was the minimal degradation product. Removing the extra fragment from the GH module led to the inability of the mutant (rAgaG4-T57) to degrade neoagarooctaose, and the final degradation products of agarose by the truncated protein were neoagarotetraose, neoagarohexaose, and neoagarooctaose at a final molar ratio of 2.7:2.8:1. The optimal temperature for agarose degradation also decreased to 40 °C for this mutant. Bioinformatic analysis suggested that tyrosine 276 within the extra fragment was a candidate active site residue for the enzymatic activity. Site-swapping experiments of Tyr(276) to 19 various other amino acids demonstrated that the characteristics of this residue were crucial for the AgaG4 degradation of agarose and the cleavage pattern of substrate.

Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.

Mfc1 Is a Novel Forespore Membrane Copper Transporter in Meiotic and Sporulating Cells

To gain insight in the molecular basis of copper homeostasis during meiosis, we have used DNA microarrays to analyze meiotic gene expression in the model yeast Schizosaccharomyces pombe. Profiling data identified a novel meiosis-specific gene, termed mfc1(+), that encodes a putative major facilitator superfamily-type transporter. Although Mfc1 does not exhibit any significant sequence homology with the copper permease Ctr4, it contains four putative copper-binding motifs that are typically found in members of the copper transporter family of copper transporters. Similarly to the ctr4(+) gene, the transcription of mfc1(+) was induced by low concentrations of copper. However, its temporal expression profile during meiosis was distinct to ctr4(+). Whereas Ctr4 was observed at the plasma membrane shortly after induction of meiosis, Mfc1 appeared later in precursor vesicles and, subsequently, at the forespore membrane of ascospores. Using the fluorescent copper-binding tracker Coppersensor-1 (CS1), labile cellular copper was primarily detected in the forespores in an mfc1(+)/mfc1(+) strain, whereas an mfc1Δ/mfc1Δ mutant exhibited an intracellular dispersed punctate distribution of labile copper ions. In addition, the copper amine oxidase Cao1, which localized primarily in the forespores of asci, was fully active in mfc1(+)/mfc1(+) cells, but its activity was drastically reduced in an mfc1Δ/mfc1Δ strain. Furthermore, our data showed that meiotic cells that express the mfc1(+) gene have a distinct developmental advantage over mfc1Δ/mfc1Δ mutant cells when copper is limiting. Taken together, the data reveal that Mfc1 serves to transport copper for accurate and timely meiotic differentiation under copper-limiting conditions.

Genomic Selection Identifies Vertebrate Transcription Factor Fezf2 Binding Sites and Target Genes

Identification of transcription factor targets is critical to understanding gene regulatory networks. Here, we uncover transcription factor binding sites and target genes employing systematic evolution of ligands by exponential enrichment (SELEX). Instead of selecting randomly synthesized DNA oligonucleotides as in most SELEX studies, we utilized zebrafish genomic DNA to isolate fragments bound by Fezf2, an evolutionarily conserved gene critical for vertebrate forebrain development. This is, to our knowledge, the first time that SELEX is applied to a vertebrate genome. Computational analysis of bound genomic fragments predicted a core consensus binding site, which identified response elements that mediated Fezf2-dependent transcription both in vitro and in vivo. Fezf2-bound fragments were enriched for conserved sequences. Surprisingly, ∼20% of these fragments overlapped well annotated protein-coding exons. Through loss of function, gain of function, and chromatin immunoprecipitation, we further identified and validated eomesa/tbr2 and lhx2b as biologically relevant target genes of Fezf2. Mutations in eomesa/tbr2 cause microcephaly in humans, whereas lhx2b is a critical regulator of cell fate and axonal targeting in the developing forebrain. These results demonstrate the feasibility of employing genomic SELEX to identify vertebrate transcription factor binding sites and target genes and reveal Fezf2 as a transcription activator and a candidate for evaluation in human microcephaly.

Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells

The solute carrier family 11 member 1 (SLC11A1) gene is strictly regulated and exclusively expressed in myeloid lineage cells. However, little is known about the transcriptional regulation of the SLC11A1 gene during myeloid development. In this study, we used HL-60 cells as a model to investigate the regulatory elements/factors involved in the transactivation of the SLC11A1 gene during phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation of HL-60 cells. Promoter deletion analysis showed that a 7-base AP-1-like element (TGACTCT) was critical for the responsiveness of the SLC11A1 promoter to PMA. Stimulation by PMA induced the binding of ATF-3 and the recruitment of two components of the SWI/SNF complex, BRG1 and β-actin, to this element in an ATF-3-dependent manner. RNAi-mediated depletion of ATF-3 or BRG1 markedly decreased SLC11A1 gene expression and its promoter activity induced by PMA. Luciferase reporter experiments demonstrated that ATF-3 cooperated with BRG1 and β-actin to activate the SLC11A1 promoter. Furthermore, we showed that PMA can induce the proximal (GT/AC)(n) repeat sequence to convert to the Z-DNA structure in the SLC11A1 gene promoter, and depletion of BRG1 resulted in a significant decrease of Z-DNA formation. Our results demonstrated that recruitment of the SWI/SNF complex initiated Z-DNA formation and subsequently helped to transactivate the SLC11A1 gene.

Electroeluting DNA Fragments

Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation.