Agarose Gel Analysis Research Articles

A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect Fasciola hepatica DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens.

Fasciola hepatica has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children and its prevalent worldwide. There is lack of data about distribution and burden of the disease in endemic regions, owing to poor efficacy of the different diagnostic methods used. A novel PCR-based test was developed by using a portable mini-PCR® platform to detect Fasciola sp. DNA and interpret the results via a fluorescence viewer and smartphone image analyzer application. Human stool, snail tissue, and water samples were used to extract DNA. Primers targeting the ITS-1 of the 18S rDNA gene of Fasciola sp. were used. The limit of detection of the mini-PCR test was 1 fg/μL for DNA samples diluted in water, 10 fg/μL for Fasciola/snail DNA scramble, and 100 fg/μL for Fasciola/stool DNA scramble. The product detection by agarose gel, direct visualization, and image analyses showed the same sensitivity. The Fh mini-PCR had a sensitivity and specificity equivalent to real-time PCR using the same specimens. Testing was also done on infected human stool and snail tissue successfully. These experiments demonstrated that Fh mini-PCR is as sensitive and specific as real time PCR but without the use of expensive equipment and laboratory facilities. Further testing of multiple specimens with natural infection will provide evidence for feasibility of deployment to resource constrained laboratories.

Heterodimeric studies of β-galactosidase genes as biocatalyst of lactose from Lactobacillus acidophilus MR-24

The current study characterized β-galactosidase-producing bacteria isolated from dairy products, in Lahore, Pakistan. Biochemical tests and the 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) hydrolysis test identified 50 Lactobacillus isolates. Fifteen isolates with blue color on X-gal plates, L. acidophilus MR-24 displayed the highest enzyme activity (905.15 U/l), characterized based on 16S rRNA gene sequencing. The enzymatic activity was enhanced 10-fold by using a Sephadex G-75 column. The highest enzyme production was obtained at pH 7.0 and a temperature of 37 °C. The β-galactosidase (lac M, L, and LM with molecular weight 951 bp, 1887 bp, and 2.8 kb respectively), was extracted from L. acidophilus MR-24. It was ligated to the pTZ57R cloning vector after Polymerase chain reaction (PCR) and agarose gel analysis. The confirmation of cloning was done via colony PCR and restriction digestion analysis. Sequencing data indicated that the enzyme consists of two overlapping regions; lac L and M encoding 70 kDa and 35 kDa protein respectively. The β-galactosidase shows significant homology with the gene from other Lactobacillus sp. Compared to crude enzymes, the co-expression of lac L and M in E. coli produced active proteins with a 30-fold increase in activity after purification via ion-exchange chromatography. The purified enzyme revealed its maximal activity at pH 7 while pH 3.0 and 9.0 showed minimal activity. The optimum temperature was 60 °C for native and 45 °C for expressed enzyme, remaining 40 % activity at 90 °C. The enzyme’s 3-dimensional (3D) structureshowed Domain N, A, and C. The L. acidophilus MR-24 strain, as a probiotic in dairy products, can provide benefits to individuals with lactose intolerance.

Comparative Study of Quercetin and Hyperoside: Antimicrobial Potential towards Food Spoilage Bacteria, Mode of Action and Molecular Docking.

The antibacterial activities of quercetin and hyperoside were evaluated towards two major spoilage bacteria in fish, Pseudomonas aeruginosa (PA) and Shewanella putrefaciens (SP). Hyperoside showed a lower minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) towards both spoilage bacteria, PA and SP, than quercetin. Cell membrane morphology was affected when treated with hyperoside and quercetin. The release of content from the treated cells occurred, as ascertained by the release of potassium and magnesium ions and the increase in conductivity of the culture media. The morphology of cells was significantly changed, in which shrinkage and pores were obtained, when observed using SEM. Both compounds negatively affected the motility, both swimming and swarming, and the formation of extracellular polymeric substance (EPS), thus confirming antibiofilm activities. Agarose gel analysis revealed that both compounds could bind to or degrade the genomic DNA of both bacteria, thereby causing bacterial death. Molecular docking indicated that the compounds interacted with the minor groove of the DNA, favoring the adenine-thymine-rich regions. Thus, both quercetin and hyperoside could serve as potential antimicrobial agents to retard the spoilage of fish or perishable products.

Chi sequences switch the RecBCD helicase–nuclease complex from degradative to replicative modes during the completion of DNA replication

Accurately completing DNA replication when two forks converge is essential to genomic stability. The RecBCD helicase-nuclease complex plays a central role in completion by promoting resection and joining of the excess DNA created when replisomes converge. chi sequences alter RecBCD activity and localize with cross-over hotspots during sexual events in bacteria, yet their functional role during chromosome replication remains unknown. Here, we use two-dimensional agarose gel analysis to show that chi induces replication on substrates containing convergent forks. The induced-replication is processive, but uncoupled with respect to leading and lagging strand synthesis, and can be suppressed by ter sites which limit replisome progression. Our observations demonstrate that convergent replisomes create a substrate that is processed by RecBCD, and that chi, when encountered, switches RecBCD from a degradative to replicative function. We propose that chi serves to functionally differentiate DNA ends created during completion, which require degradation, from those created by chromosomal double-strand breaks, which require resynthesis.

Separation Properties of Plasmid DNA Using a Two-Stage Particle Adsorption-Microfiltration Process

Plasmid DNA is used as a vector for gene therapy and DNA vaccination; therefore, the establishment of a mass production method is required. Membrane filtration is widely employed as a separation method suitable for the mass production of plasmid DNA. Furthermore, the separation of plasmid DNA using microfiltration and ultrafiltration membranes is being investigated. Because plasmid DNA has a circular structure, it undergoes significant deformation during filtration and easily permeates the membrane, hindering the selection of separation membranes based on molecular weight. In this study, we applied affinity microfiltration to plasmid DNA purification. α-Fe2O3 with an isoelectric point of approximately 8 and a particle size of 0.5 μm was selected as the ligand for two-stage affinity microfiltration of plasmid DNA. In the first stage of microfiltration, the experiment was conducted at a pH of 5, and a cake of α-Fe2O3 with bound plasmid DNA was obtained. Next, liquid permeation (pH 9 and 10) through the cake was performed to elute the bound plasmid DNA. Plasmid DNA was eluted during the early phase of liquid permeation at pH 10. Furthermore, agarose gel analysis confirmed the usefulness of the two-stage affinity microfiltration method with adsorption and desorption for plasmid DNA purification.

Real-Time PCR Method for Assessment of ParA-Mediated Recombination Efficiency in Minicircle Production.

The in vivo intramolecular recombination of a parental plasmid allows excising prokaryotic backbone from the eukaryotic cassette of interest, leading to the formation of, respectively, a miniplasmid and a minicircle. Here we describe a real-time PCR protocol suitable for the determination of recombination efficiency of parental plasmids with multimer resolution sites (MRS). The protocol was successfully applied to purified DNA samples obtained from E. coli cultures, allowing a more reproducible determination of recombination efficiency than densitometry analysis of agarose gels.

Nucleic acid extraction from complex biofluid using toothpick-actuated over-the-counter medical-grade cotton

Nucleic acid amplification technique (NAAT)-assisted detection is the primary intervention for pathogen molecular diagnostics. However, NAATs such as quantitative real-time polymerase chain reaction (qPCR) require prior purification or extraction of target nucleic acid from the sample of interest since the latter often contains polymerase inhibitors. Similarly, genetic disease screening is also reliant on the successful extraction of pure patient genomic DNA from the clinical sample. However, such extraction techniques traditionally utilize spin-column techniques that in turn require centralized high-speed centrifuges. This hinders any potential deployment of qPCR- or PCR-like NAAT methods in resource-constrained settings. The development of instrument-free nucleic acid extraction methods, especially those utilizing readily available materials would be of great interest and benefit to NAAT-mediated molecular diagnosis workflows in resource-constrained settings. In this report, we screened medical-grade cotton, a readily available over-the-counter biomaterial to extract genomic DNA (gDNA) spiked in 30 %, 45 %, and 60 % serum or cell lysate. The extraction was carried out in a completely instrument-free manner using cotton and a sterilized toothpick and was completed in 30 min (with using chaotropic salt) or 10 min (without using chaotropic salt). The quality of the extracted DNA was then probed using PCR followed by agarose gel analysis for preliminary validation of the study. The qPCR experiments then quantitatively established the extraction efficiency (0.3–27 %, depending on serum composition). Besides, percent similarity score obtained from the Sanger sequencing experiments probed the feasibility of extracted DNA towards polymerase amplification with fluorescent nucleotide incorporation. Overall, our method demonstrated that DNA extraction could be performed utilizing toothpick-mounted cotton both with or without using a chaotropic salt, albeit with a difference in the quality of the extracted DNA.

Proteomic and phosphoproteomic profiling in heart failure with preserved ejection fraction (HFpEF).

Although the prevalence of heart failure with preserved ejection fraction (HFpEF) is increasing, evidence-based therapies for HFpEF remain limited, likely due to an incomplete understanding of this disease. This study sought to identify the cardiac-specific features of protein and phosphoprotein changes in a murine model of HFpEF using mass spectrometry. HFpEF mice demonstrated moderate hypertension, left ventricle (LV) hypertrophy, lung congestion and diastolic dysfunction. Proteomics analysis of the LV tissue showed that 897 proteins were differentially expressed between HFpEF and Sham mice. We observed abundant changes in sarcomeric proteins, mitochondrial-related proteins, and NAD-dependent protein deacetylase sirtuin-3 (SIRT3). Upregulated pathways by GSEA analysis were related to immune modulation and muscle contraction, while downregulated pathways were predominantly related to mitochondrial metabolism. Western blot analysis validated SIRT3 downregulated cardiac expression in HFpEF vs. Sham (0.8 ± 0.0 vs. 1.0 ± 0.0; P < 0.001). Phosphoproteomics analysis showed that 72 phosphosites were differentially regulated between HFpEF and Sham LV. Aberrant phosphorylation patterns mostly occurred in sarcomere proteins and nuclear-localized proteins associated with contractile dysfunction and cardiac hypertrophy. Seven aberrant phosphosites were observed at the z-disk binding region of titin. Additional agarose gel analysis showed that while total titin cardiac expression remained unaltered, its stiffer N2B isoform was significantly increased in HFpEF vs. Sham (0.144 ± 0.01 vs. 0.127 ± 0.01; P < 0.05). In summary, this study demonstrates marked changes in proteins related to mitochondrial metabolism and the cardiac contractile apparatus in HFpEF. We propose that SIRT3 may play a role in perpetuating these changes and may be a target for drug development in HFpEF.

Serum Protein Electrophoresis Reference Intervals for Six Species of Wild-Sampled Sharks in South Florida.

Serum proteins found in the blood have been used as diagnostic markers in both human and animal medicine. Evaluating proteins in the blood of sharks may be a valuable way to assess shark health. The purpose of this study was to perform serum protein electrophoresis from non-lethal blood samples of wild sharks and establish baseline values for six species off South Florida: blacknose (Carcharhinus acronotus), blacktip (C. limbatus), bull (C. leucas), lemon (Negaprion brevirostris), sandbar (C. plumbeus), and tiger shark (Galeocerdo cuvier). Two hundred and fifty-one serum samples were collected between 2015-2018 along with sex determination and morphometric measurements. Agarose gel analysis was performed using mammalian protein fraction delimits albumin, alpha 1-globulins, alpha 2-globulins, beta globulins, and gamma globulins alternatively defined as fractions 1-5. Reference electrophoretic profiles were established for each species, revealing the dominance of protein fractions 3 and 4. The fraction 3:4 ratio was determined for each shark and reference intervals for total solids, protein fractions, and fraction 3:4 ratio are reported for each species. There were no significant differences in total solids or protein fractions between males and females of any species. In both blacktip sharks and bull sharks, total solids were positively correlated with body size, but not in the other species evaluated. In blacknose sharks, the fraction 3:4 ratio was positively correlated with body condition. Results suggest that there may be differences in total solids values across dry and wet seasons for some species. Newly established reference intervals can now be utilized in future research to evaluate the health of captive sharks and to investigate the health status of individuals in the wild as well as relate health measures to environmental conditions.

Detection of fimH virulence gene in uropathogenic Escherichia coli Using PCR

Urinary tract infections (UTIs) are one of main health problems caused by many microorganisms, including uropathogenic Escherichia coli (UPEC). The virulence factors are important in colonization of UPEC, extra-intestinal survival, and creation of cytopathic effects. In addition, the expression of special virulence factors of UPEC can contribute to uropathogenicity, as well as worsening of UTIs. In this study, 134 isolates of uropathogenic E. coli were collected from Baghdad/Iraq through March 2018 to April 2019. UPEC were identified by culturing on Hardy chromo UTI agar and confirmed at molecular level by using uidA housekeeping gene using real time polymerase chain reaction.The prevalence of fimH gene was studied among 134uropathogenic Escherichia coli of patients attended Using polymerase chain reaction assay, Visualization of fimH gene (bp) by 1.5% agarose gel analysis. By comparing the bands in a sample to the DNA ladder, their approximate sizes can be determining. For instance, the bright band on the gel above 1000 base pairs (bp) in size for fimH. The intended gene was detected in 94.8% of the isolates and absence in 5.2%.of them.

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

Recombination efficiency measurement by real-time PCR: A strategy to evaluate ParA-mediated minicircle production

Minicircles (MCs) are DNA molecules that are produced in Escherichia coli by replicating a parental plasmid (PP) and inducing its site-specific intramolecular recombination into miniplasmid (MP; containing the prokaryotic backbone) and MC molecules (comprised by the eukaryotic cassette). The determination of the recombination efficiency and the monitoring of PP, MC and MP species during processing and in the final product are critical aspects of MC manufacturing. This work describes a real-time PCR method for the specific identification of PP, MP or MC that uses sets of primers specific for each species. The method was evaluated using artificial mixtures of (i) PP and MP, (ii) PP and MC and (iii) MP and MC that were probed for all three DNA molecules. The ratio of molecules of each DNA species in these mixtures were determined with differences lower than 10% relatively to the expected ratio of the species in 90% of the mixtures. Next, the recombination efficiency was successfully estimated by analysing pre-purified DNA samples obtained from cell cultures. A standard deviation < 2% was obtained between replicas and results closely correlated with those obtained by densitometry analysis of agarose gels. Further optimization is required to determine recombination efficiency directly from whole cells.

Duckweed Species Genotyping and Interspecific Hybrid Discovery by Tubulin-Based Polymorphism Fingerprinting.

Duckweeds (Lemnaceae) are the smallest and fastest-growing angiosperms. This feature, together with high starch production and good nutritional properties, makes them suitable for several applications, including wastewater treatment, bioenergy production, or feed and food supplement. Due to their reduced morphology and great similarity between diverse species, taxonomic identification of duckweeds is a challenging issue even for experts. Among molecular genotyping methods, DNA barcoding is the most useful tool for species identification without a need for cluster analysis. The combination of two plastid barcoding loci is now considered the gold standard for duckweed classification. However, not all species can be defined with confidence by these markers, and a fast identification method able to solve doubtful cases is missing. Here we show the potential of tubulin-based polymorphism (TBP), a molecular marker based on the intron length polymorphisms of β-tubulin loci, in the genomic profiling of the genera Spirodela, Landoltia, and Lemna. Ninety-four clones were analyzed, including at least two representatives of each species of the three genera, with a special focus on the very heterogeneous species Lemna minor. We showed that a single PCR amplification with universal primers, followed by agarose gel analysis, was able to provide distinctive fingerprinting profiles for 10 out of 15 species. Cluster analysis of capillary electrophoresis–TBP data provided good separation for the remaining species, although the relationship between L. minor and Lemna japonica was not fully resolved. However, an accurate comparison of TBP profiles provided evidence for the unexpected existence of intraspecific hybrids between Lemna turionifera and L. minor, as further confirmed by amplified fragment length polymorphism and sequence analysis of a specific β-tubulin locus. Such hybrids could possibly correspond to L. japonica, as originally suggested by E. Landolt. The discovery of interspecific hybrids opens a new perspective to understand the speciation mechanisms in the family of duckweeds.

Analysis of protein denaturation, aggregation and post-translational modification by agarose native gel electrophoresis

Agarose native gel electrophoresis has been developed to separate proteins and protein complexes in the native state. Here, we applied this technology to analyze proteins that undergo degradation, post-translational modification or chemical/physical changes. Antibodies showed aggregation/association upon acid or heat treatment. Limited reduction of disulfide bonds resulted in non-covalent aggregation of bovine serum albumin and cleavage of only inter-chain linkages of an antibody that had no effects on its overall structure. Native agarose gel analysis showed changes in mobility of human transferrin upon Fe3+ binding. Analysis of a commercial glycated human hemoglobin A1c showed no difference in electrophoretic pattern from un-modified hemoglobin. Native agarose gel showed aggregation of a virus upon acid or heat treatment. We have extracted bands of bovine serum albumin from the agarose native gel for sodium dodecylsulfate gel electrophoresis analysis, showing degradation of aged sample. Lastly, we analyzed phosphorylation of Zap70 kinase by native gel and Western blotting. These applications should expand the utility of this native gel electrophoresis technology.

A Fluorescent Cell-Based Technique for Monitoring Efflux of MRP4

Background: Overexpression of efflux pumps is the drug resistance and adaptation mechanism employed by some eukaryotes and bacteria to transport endogenous and chemotherapeutic compounds from the intracellular to the extracellular environment. Aim: The study aimed at establishing a fluorescent cell-based assay to monitor the efflux activities of an ABC-transporter, multi-drug resistance protein 4 (MRP4). Methods: DH5α competent E. coli cells were transformed with pcDNA-MRP4 by the heat-shock process. The presence of the MRP4 gene was analyzed by the digestion of plasmid using EcoRI and analyzed on a 1% agarose gel. HEK 293 cells were transfected with purified pcDNA-MRP4 under optimized conditions using a Polyethylenimine (PEI) protocol. The level of MRP4 in the HEK 293 cells was characterized by western blotting analysis using M4I-10 anti-MRP4 and anti-Rat IgG (whole molecule)-Alkaline phosphatase antibodies. The fluorescent uptake study was performed by the incubation of 0.02 mM 8-[fluo-cAMP] with the MRP4-transfected and control HEK 293 cells for 1 h. The level of fluorescence was analyzed using fluorescence microscopy and spectrometer. Results: The agarose gel analysis showed a plasmid of 9.4 kb and restriction product of 5 kb, which correspond with the pcDNA and MRP4 sizes respectively. The western blot results of the transfection showed 4 μg pcDNA-MRP4 and the N/P ratio of 9 was the optimized condition to transfect our HEK 293 cells as it showed the broadest band. In the efflux studies, the fluorescence images of the MRP4-transfected HEK 293 cells were very low compared to the untransfected control. The level of fluorescence accumulation was significantly (P ≤ 0.0001) higher 228.6 ± 13.1 RFU in the untransfected cells than the MRP4-transfected cells 8.6 ± 1.8 RFU. Conclusion: The higher levels of fluorescence detected in the control in both the fluorescent microscopy and spectrophotometer showed that MRP4-transfected cells had effluxed the 8-[fluo-cAMP] substrate out of the cell. This method could be employed in the detection of MRP4 functions in bacteria and cancer cells.

Strategies for the Reconstitution and Purification of Haloarchael Protein RadA

DNA repair proteins in halophilic organisms are interesting to study in the context of understanding the dynamics of protein-DNA interaction and their adaptation to perform biochemical activities at high osmolarity. Successful expression and purification of halophilic proteins is often challenging particularly when they are over-expressed in non-halophilic heterologous host. In the present study, radA from Haloferax volcanii was cloned and overexpressed in E. coli. Although, radA was over-expressed as a soluble protein in E. coli but purification of RadA seemed challenging. Various strategies were therefore implemented to attain maximum possible purification of RadA. The purification of RadA using Immobilized Metal Affinity Chromatography (IMAC) followed by Size Exclusion Chromatography (SEC) was initially adopted. The SDS-PAGE and agarose gel analysis of the representative fractions from SEC indicated this to be an unsuccessful strategy due to high affinity of protein with the DNA from host. The refolding strategy employing denaturation of the RadA in urea along with benzonase treatment was attempted to chop down the contaminating host DNA. This was observed an effective method as the subsequent analysis of the representative fractions from SEC indicated RadA at about 90% purity that can possibly be suitable for further biochemical and structural analysis.

Ion-induced PCR strategy for mercury detection.

Mercury contamination is one of the most serious environmental problems. It can cause serious effects on the human health, such as case damage in the brain, nervous system, immune system, and kidney failure. Therefore, development of an accurate, sensitive, and simple operational detection method for mercury is very necessary. Herein, we report a new strategy for mercury ion detection based on commonly used PCR technique. High selectivity and sensitivity were achieved by the formation of the thymine-Hg-thymine (T-Hg-T) unnatural base pair at the 3'-end of PCR primers. The detection results of PCR amplification in presence of mercury ion could be reported either by using agarose gel analysis or through real-time fluorometric dye tracing for different detection purposes. To our knowledge, this study represents the first application of PCR based technique to the detection of metal ions.

Isolation of Partial Housekeeping Genes on Tuntun Angin (Elaeocarpus floribundu)

Some genes like 18S rRNA, 26S rRNA, elongation factor 1-alpha (EF1a), and beta-tubulin (TUB) are members of housekeeping genes group that are commonly used as internal control in gene expression study. This study aimed to isolate those four housekeeping genes of tuntun angin (Elaeocarpus floribundus). The research material included fresh leaves of E. floribundus that were picked up from Kajuik Lake in Riau Province and four primer pairs. The procedures consisted of total DNA isolation using Genomic DNA Mini Kit Plant (Geneaid), polymerase chain reaction (PCR), electrophoresis on 1% agarose gel, sequencing, and bioinformatic analysis. This study has been isolated 18S rRNA, 26S rRNA, EF1a, and TUB genes with the size of 422 bp, 922 bp, 856 bp, and 877 bp, respectively. The EF1a and TUB genes has never been reported in Elaeocarpaceae family. Thus, those partial DNA sequences are the first sequences reported from this species and can be used as reference genes in this plant after validation.

PTB-AS, a Novel Natural Antisense Transcript, Promotes Glioma Progression by Improving PTBP1 mRNA Stability with SND1.

Glioma, the most common primary malignancy in the brain, has high recurrence and lethality rates, and thus, elucidation of the molecular mechanisms of this incurable disease is urgently needed. Poly-pyrimidine tract binding protein (PTBP1, also known as hnRNP I), an RNA-binding protein, has various mechanisms to promote gliomagenesis. However, the mechanisms regulating PTBP1 expression are unclear. Herein, we report a novel natural antisense noncoding RNA, PTB-AS, whose expression correlated positively with PTBP1 mRNA. We found that PTB-AS significantly promoted the proliferation and migration invivo and invitro of glioma cells. PTB-AS substantially increased the PTBP1 level by directly binding to its 3' UTR and stabilizing the mRNA. Furthermore, staphylococcal nuclease domain-containing 1 (SND1) dramatically increased the binding capacity between PTB-AS and PTBP1 mRNA. Mechanistically, PTB-AS could mask the binding site of miR-9 in the PTBP1-3' UTR; miR-9 negatively regulates PTBP1. To summarize, we revealed that PTB-AS, which maintains the PTBP1 level through extended base pairing to the PTBP1 3' UTR with the assistance of SND1, could significantly promote gliomagenesis.

Integration of Authentic Research into an Undergraduate Laboratory Course: Design and Synthesis of a Gene Therapy Vector

Undergraduate Biology students often graduate without exposure to authentic research experiences. Laboratory courses often follow a one or two week fail‐proof experiment resembling a cookbook recipe, lacking the uncertainty of genuine research. Techniques in genetics and molecular biology cover a wide array of skills that are essential to succeed in a biotechnological laboratory today. Can we design a lab course based on the teaching of concepts while imparting the skills and applications of modern techniques to undergraduate students? In our experimental laboratory course, we focus on the implementation of genetics and molecular biology lab techniques, thus providing students with theoretical concepts and laboratory skills. We prepare students to carry‐out scientific protocols that can be applied to a future workforce setting. Students are immersed in an 11‐week series of labs with the objective to use molecular cloning to make a gene therapy vector; therapies are designed to inhibit the overexpression of oncogenes in brain tumors. Students in the laboratory are introduced to PubMed and Genbank to research the background of the target gene. Students use DNA analysis software, Serial Cloner, as an interactive tool to evaluate DNA sequences and visualize therapy design, with each student generating their own therapy vector. Students isolate and purify DNA using a plasmid purification protocol, and measure quantity and quality using a spectrophotometer. They are introduced to molecular cloning and the value of master mixes, beginning with the digestion of their DNA plasmid using restriction enzymes. Students, making their own buffers and gels, perform gel electrophoresis and subsequently cut‐out the appropriate bands. Students purify bands and clean‐up DNA with magnetic beads, preparing samples for the next step. Students perform DNA ligation with proper controls and transform ligations into DH5‐α super competent cells. To verify successful transformation, students make Luria Broth ampicillin plates and spread transformed bacteria onto plates; ligation efficiency is measured based on bacterial colony count. The students then use colony PCR and agarose gel analysis to verify successful ligations. At the end of the semester, students compile results from each step during the 11‐week lab series and integrate learning from the lecture to synthesize a comprehensive lab report. Student evaluation is based on connecting lecture concepts to laboratory experiments, including clearly stating lab objectives, writing protocols in own words, accurately recording results in notebook (in pen), labeling of figures, analysis of data, and comprehension of results. Properly recording and understanding results is more highly valued over expected results. Successful experiments carried‐out in the course are used to contribute to authentic research projects. The goal of this lab course is to integrate authentic research into the undergraduate learning experience, preparing students for graduate school and employment in a modern biotechnological lab; establishing the next generation of scientist.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.