Purified Plasmid DNA Research Articles

Escherichia coli persisters in biofilm can perform horizontal gene transfer by transformation

Persisters represent a subset of cells that exhibit transient tolerance to antimicrobials. These persisters can withstand sudden exposure to antimicrobials, even as the majority of normal cells perish. In this study, we have demonstrated the capacity of ampicillin-tolerant and alkali-tolerant persisters to execute horizontal gene transfer via in situ transformation within biofilms. Air–solid biofilms, comprising two Escherichia coli populations each with a distinct plasmid, were formed on agar media. They were treated with lethal doses of ampicillin or NaOH for 24 h, followed by a 1-min glass-ball roll. This process led to a high frequency of horizontal plasmid transfer (10−7–10−6 per cell) from dead cells to surviving persisters within the biofilms. Plasmid transfer was DNase-sensitive and also occurred by adding purified plasmid DNA to plasmid-free biofilms, demonstrating a transformation mechanism. This marks the first evidence of persisters’ novel ability for horizontal gene transfer, via transformation.

A project‐based learning mode for science students: Fluorescent protein expression in trypanosomes

AbstractAn effective, practical approach for incorporating molecular biology tools to biology and biochemistry students is described. For 6 weeks, students are exposed to bacterial transformation, plasmid DNA purification and analysis, parasite transfection, ectopic expression of fluorescent proteins, fluorescence microscopy, and flow cytometry analysis. This research‐oriented laboratory course delves into theoretical and practical concepts of basic techniques in molecular biology to emphasize the logical connections between the obtained results and the resolution of problems that arise daily in a research laboratory, training the students in planning, conducting, discussing, and presenting their own experiments and results.

Grafting chromatographic monoliths with charged linear polymers for highly productive and selective protein or plasmid DNA purification

Increased requirements for plasmid DNA (pDNA) in gene therapy and vaccination efforts bring the need for efficient large-scale production processes with high purity and productivity. Chromatographic purification of pDNA is often a bottleneck due to low dynamic binding capacities (DBC), pDNA recovery and/or low selectivity of columns. Ion exchangers (IEX) with charged groups on surface extenders (polyions formed by a grafting reaction and subsequents modification) have attracted attention due to their greatly enhanced productivity compared with traditional IEX. The ultimate goal of our investigation was to increase the DBC for pDNA on a monolith stationary phase by grafting the surface with linear polymethacrylate brushes (grafted chains), while retaining high pDNA recovery and chromatographic selectivity between pDNA and RNA impurities. The inner walls of the Convective Interaction Media® monolith channels were successfully coated with polyglycidyl methacrylate linear chains of varying densities and lengths by employing a controlled radical polymerization reaction. Glycidyl groups throughout the length of the formed grafted chains were converted to cation- (CEX) or anion-exchanging (AEX) moieties. Preliminary chromatographic evaluation of novel grafted CEX columns for different model proteins exhibited 10-times higher DBC with high elution recovery (99 %). Similar DBC improvement was proven for 7.3 kilobase pair pDNA, where the DBC10 increased from 1.8 mg pDNA per mL of non-grafted chromatographic support up to 15.2 mg per the most densely distributed and the longest grafted chains, modified with weak AEX groups. However, the DBC increase was partially at the expense of pDNA elution recovery, which presumably decreased due to entanglement of pDNA molecules inside the dense grafted layer. To confirm the hypothesis, we have prepared a series of columns differing in length and density of grafted chains and tested them for pDNA capture. A satisfactory compromise between high pDNA DBC and elution recovery was found with relatively long and low density grafted chains. The optimal grafted AEX column with 1 mL bed volume was evaluated for pDNA capture from neutralized E. coli lysate. With a capacity of 13.5 mg pDNA per mL support, ≥95 % elution recovery, and complete RNA clearance, the pDNA was successfully purified at loading flow rate of at least 15 column volumes per min. According to our knowledge and literature search, these process characteristics would enable one of the highest pDNA isolation productivities to be achieved with currently tested IEX.

The Construction of CRISPR-Cas9 System Targeting Vector for CYP3A4 Gene in Hepatic Cell Lines

Drug metabolism in the human liver initiated by Cytochrome P450 (CYP450) has been widely acknowledged. They oxidise drugs, harmful compounds, and endogenous molecules like steroids. In our research study, we mainly focused on CYP3A4 of the CYP450 subfamily that is widely available in the liver around 15% - 20% of hepatic content and plays a significant function in metabolising up to 50% of drugs in the market today. However, the CYP3A4 gene encodes CYP3A4 enzymes that are highly polymorphic, which could affect enzymatic activity and cause variable responses in drug metabolism. Clustered Regularly Interspaced Short Palindromic Repeats Associated Protein 9 also known as the CRISPR-Cas9 system is a gene editing method that allows scientists to edit parts of the genome by insertions and deletions. Hence, this method would be useful for genetic variants such as the CYP3A4 gene. The objectives of this study are to design a guide RNA (gRNA), to insert the designed gRNA into pGuide-it-Zs-Green1 vector, to quantify the purified CYP3A4-KO plasmid vector by using NanoDrop® and to transfect the constructed vector in a hepatic cell line. The methodology involved the selection of the gRNA by using the online gene editing tool, Synthego (https://design.synthego.com), annealing oligos of the gRNA for CYP3A4, cloning gRNA into a plasmid vector, isolation, and purification of the CYP3A4-KO plasmid vector. The construction of the CRISPR-Cas9 targeting vector in this study was successfully achieved and promising since the selected gRNA for CYP3A4 gene which is 5’-ATAAATCCCACTGGACCAAA-3’ and located in exon 5 was correctly ligated after the confirmed with sequencing reaction and cloned it into a plasmid vector. The yield of pCYP3A4-KO plasmid DNA was a good candidate for transfection.

MOLECULAR CHARACTERIZATION OF ANAPLASMA PHAGOCYTOPHILUM IN IXODID TICKS IN KAYSERİ REGION IN TURKEY

The study was conducted to investigate the presence of Anaplasma phagocytophilum in ixodid ticks collected from cattle in Kayseri region of Turkey using Real Time PCR and to characterize positive isolates based on 16S rRNA gene region. DNA was extracted from 265 adult ticks. Nested PCR analyses were performed using Anaplasma phagocytophilum-specific primers amplifying a 641 bp fragment of 16S rRNA gene region. Real Time PCR analysis revealed positive results in one H. marginatum and R. turanicus sample. DNA sequences were submitted to GenBank and analyzed by pairwise and multiple sequence alignments with other A. phagocytophilum strains in GenBank to investigate the phylogeny. The phylogenetic analysis revealed that A. phagocytophilum isolates collected from H. marginatum and R. turanicus samples in Kayseri region clustered into three main groups (A, B, and C) with previously reported isolates from the world. A and B groups showed high homology, whereas C group had an average genetic variation of 0.2%. The average genetic differences between A and B groups were 10.8±2.0 and 13.0±2.7% between A and C groups, while the average genetic difference between B and C groups was 13.8±2.8%. In conclusion, this study provides scientific data on molecular prevalence and genetic characteristics of A. phagocytophilum in tick samples in Turkey.

A scalable single-use two-step plasmid purification process

A scalable single-use two-step plasmid purification process

Purification of linearized template plasmid DNA decreases double-stranded RNA formation during IVT reaction.

After the COVID-19 pandemic, messenger RNA (mRNA) has revolutionized traditional vaccine manufacturing. With the increasing number of RNA-based therapeutics, valuable new scientific insights into these molecules have emerged. One fascinating area of study is the formation of double-stranded RNA (dsRNA) during in vitro transcription (IVT) which is considered a significant impurity, as it has been identified as a major trigger in the cellular immune response pathway. Therefore, there is a growing importance placed to develop and optimize purification processes for the removal of this by-product. Traditionally, efforts have primarily focused on mRNA purification after IVT through chromatographic separations, with anion exchange and reverse phase chromatography emerging as effective tools for this purpose. However, to the best of our knowledge, the influence and significance of the quality of the linearized plasmid have not been thoroughly investigated. Plasmids production involves the growth of bacterial cultures, bacterial harvesting and lysis, and multiple filtration steps for plasmid DNA purification. The inherent complexity of these molecules, along with the multitude of purification steps involved in their processing, including the subsequent linearization and the less-developed purification techniques for linearized plasmids, often result in inconsistent batches with limited control over by-products such as dsRNA. This study aims to demonstrate how the purification process employed for linearized plasmids can impact the formation of dsRNA. Several techniques for the purification of linearized plasmids based on both, resin filtration and chromatographic separations, have been studied. As a result of that, we have optimized a chromatographic method for purifying linearized plasmids using monolithic columns with C4 chemistry (butyl chains located in the surface of the particles), which has proven successful for mRNAs of various sizes. This chromatographic separation facilitates the generation of homogeneous linearized plasmids, leading to mRNA batches with lower levels of dsRNA during subsequent IVT processes. This finding reveals that dsRNA formation is influenced not only by RNA polymerase and IVT conditions but also by the quality of the linearized template. The results suggest that plasmid impurities may contribute to the production of dsRNA by providing additional templates that can be transcribed into sequences that anneal with the mRNA molecules. This highlights the importance of considering the quality of plasmid purification in relation to dsRNA generation during transcription. Further investigation is needed to fully understand the mechanisms and implications of plasmid-derived dsRNA. This discovery could shift the focus in mRNA vaccine production, placing more emphasis on the purification of linearized plasmids and potentially saving, in some instances, a purification step for mRNA following IVT.

Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses.

The continued acceleration of time-to-market product development and rising demand for biotherapeutics have hastened the need for higher throughput within the biopharmaceutical industry. Automated liquid handlers (ALH) are increasingly popular due to flexible programming that enables processing of multiple samples with an array of functions. This flexibility is useful in streamlining research that requires chromatographic procedures to achieve product purity for downstream analysis. However, purification of biologics often requires additional off-deck buffer exchange steps due to undesirable elution conditions such as high acid or high salt content. Expanding the capability of ALHs to perform purification in sequence with buffer exchange would, therefore, increase workflow efficiency by eliminating the need for manual intervention, thus expediting sample preparation. Here we demonstrate two different automated purifications using pipet-based dispersive solid-phase extraction (dSPE). The first is an affinity purification of His-tagged proteins from bacterial lysate. The second is an anion-exchange purification of plasmid DNA. Both methods are followed by buffer exchange performed by an ALH. Percent recoveries for the three purified recombinant proteins ranged from 51 ± 1.2 to 86 ± 10%. The yields were inversely correlated to starting sample load and protein molecular weight. Yields for plasmid purification ranged between 11.4 ± 0.8 and 13.7 ± 0.9 µg, with the largest plasmid providing the highest yield. Both programs were rapid, with protein purification taking <80 minutes and plasmid purification <60 minutes. Our results demonstrate that high-quality, ready-to-use biologics can be obtained rapidly from a crude sample after two separate chromatographic processes without manual intervention.

Purification of a recombinant oncolytic virus from clarified cell culture media by anion exchange monolith chromatography.

The use of viral vectors for vaccine, gene therapy, and oncolytic virotherapy applications has received increased attention in recent years. Large-scale purification of viral vector-based biotherapeutics still presents a significant technical challenge. Chromatography is the primary tool for the purification of biomolecules in the biotechnology industry; however, the majority of chromatography resins currently available have been designed for the purification of proteins. In contrast, convective interaction media monoliths are chromatographic supports that have been designed and successfully utilized for the purification of large biomolecules, including viruses, viruslike particles, and plasmids. We present a case study on the development of a purification method for recombinant Newcastle disease virus directly from clarified cell culture media using strong anion exchange monolith technology (CIMmultus QA, BIA Separations). Resin screening studies showed at least 10 times higher dynamic binding capacity of CIMmultus QA compared to traditional anion exchange chromatography resins. Design of experiments was used to demonstrate a robust operating window for the purification of recombinant virus directly from clarified cell culture without any further pH or conductivity adjustment of the load material. The capture step was successfully scaled up from 1mL CIMmultus QA columns to the 8L column scale and achieved a greater than 30-fold reduction in process volume. Compared to the load material, total host cell proteins were reduced by more than 76%, and residual host cell DNA by more than 57% in the elution pool, respectively. Direct loading of clarified cell culture onto a high-capacity monolith stationary phase makes convective flow chromatography an attractive alternative to centrifugation or TFF-based virus purification procedures.

Effect of plasmid DNA isoforms on preparative anion exchange chromatography.

Increased need for plasmid DNA (pDNA) with sizes above 10kbp (large pDNA) in gene therapy and vaccination brings the need for its large-scale production with high purity. Chromatographic purification of large pDNA is often challenging due to low process yields and column clogging, especially using anion-exchanging columns. The goal of our investigation was to evaluate the mass balance and pDNA isoform composition at column outlet for plasmids of different sizes in combination with weak anion exchange (AEX) monolith columns of varying channel size (2, 3 and 6µm channel size). We have proven that open circular pDNA (OC pDNA) isoform is an important driver of reduced chromatographic performance in AEX chromatography. The main reason for the behaviour is the entrapment of OC pDNA in chromatographic supports with smaller channel sizes. Entrapment of individual isoforms was characterised for porous beads and convective monolithic columns. Convective entrapment of OC pDNA isoform was confirmed on both types of stationary phases. Porous beads in addition showed a reduced recovery of supercoiled pDNA (on an 11.6kbp plasmid) caused by diffusional entrapment within the porous structure. Use of convective AEX monoliths or membranes with channel diameter >3.5µm has been shown to increase yields and prevent irreversible pressure build-up and column clogging during purification of plasmids at least up to 16kbp in size.

Extraction &amp; Purification of Plasmid from Some Spices of E. coli in Different Ways

This study involved the use of multiple methods to separate plasmids from bacterial cell DNA for some isolates of pathogenic E. coli through several steps, starting with the analysis of the bacterial cell using lysozymes to remove the outer wall, followed by centrifugation to isolate plasmids found in the solution from the rest of the proteins and other forms of DNA. Many sequential methods were used to separate plasmids. The first method used was the basal denaturation sodium hydroxide-based, which led to the denaturation of the chromosomal DNA without affecting the plasmid DNA, followed by the addition of sodium acetate, which led to the preservation of the shape and structure of the plasmid DNA. Second, using cesium chloride gradient density to isolate the protein cell components and the rest of the DNA forms. The different densities of these components led to the appearance of sequential bundles depending on their different molecular weights. Ethidium bromide, which gave the plasmid bundles a fluorescent dye, was added using ultraviolet rays. The last purification method was using the boiling method using a water bath. Plasmid samples extracted from the previous methods were taken to perform the purification and separation process using the high electrophoresis method. Akarose gel was used to separate the high molecular weight protein fragments. Standard proteins and plasmids were migrated to determine the volumes of purified plasmids.

Optimization of Biolistic Transformation Parameters for &lt;i&gt;Nicotiana tabacum&lt;/i&gt;

Biolistics is one of the widely used methods to deliver nucleic acids into plant cells. In this work, we optimized the protocol for biolistic transformation of Nicotiana tabacum with the gene gun PDS-1000/He Biolistic Particle Delivery System. The Green Fluorescent Protein (GFP) gene was used as a marker. The optimal parameters for the transformation of N. tabacum leaf cells were determined as: pressure, 1350 psi; tungsten particle size, 1.3 μm; plasmid DNA purification method, ethanol re-precipitation. The results should be useful for the development of biolistic transformation protocols for plant cells, including application for plant genome editing of agricultural species.

Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device

BackgroundSequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications.ResultsHere, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies’ MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 to 0.53%, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100% accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing.ConclusionsOur pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.

RECOVERING AND STANDARDIZING OF THE ORIGINAL GENOTHERAPEUTIC CONSTRUCTION FOR STIMULATION OF NERVOUS TISSUE REGENERATION

Traumatic nerve injury is one of the most common cause of permanent disability. The problem of nerve regeneration is based on the slow growth of nerve fibers and short-term production of neutrophic factors (NGF, BDNF, NT-3, GDNF, etc.), which stimulate the survival of damaged neurons and growth of nerve fibers. In most cases, the intrinsic regenerative potential is insufficient to restore innervation, which requires the replacement therapy with neurotrophins. One of the most promising therapeutic approaches for regenerative medicine is gene therapy that allows to prolong the local production of neurotrophic factors. Previously, we created a bicistronic gene therapy construct pNCure (a plasmid for the treatment of nerves), based on the modified pVax1 plasmid construct approved by the FDA (U.S. Food and Drug Administration), and encoding the cDNA sequences of brain-derived neurotrophic factor (BDNF) and urokinase-type plasminogen activator (uPA). It revealed a prominent therapeutic activity in a model of traumatic nerve injury in mice. Gene therapy products is a particular class of drugs that need particular approaches to production, purification and standartization. There is practically no information on the purification and standardization of gene therapy drugs in Russian, and this is the topic of our manuscript. Here, we have elucidat-ed the issues of plasmid production in E. coli cells with subsequent multistage purification, as well as the issues of evaluation the identity and quantity of the plasmid (using PCR and UV spectrometry methods), and determination of pH and quantity of related and secondary impurities within the sub-stance. The results of the study have demonstrated that the proposed approach to plasmid production, purification and standartization allows to obtain the desired high-quality gene therapy drug that meets all the requirements of the XIV State Pharmacopia.

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.

Plasmid Profile and Enterobacterial Repetitive Intergenic Consensus-PCR Characterization of Salmonella Infantis Isolates Recovered From Poultry Sources

Background: Salmonella is known as one of the most important bacterial agents infecting both humans and animals. Salmonella Infantis has been reported as one of the 15 most prevalent serovars worldwide. Despite its clinical importance, there is little information on the molecular characteristics of S. Infantis in Iran. Objectives: This study was conducted to characterize S. Infantis isolates collected from poultry sources in the last decade. The isolates were typified by plasmid profile and enterobacterial repetitive intergenic consensus (ERIC-PCR). Methods: Forty S. Infantis isolates from poultry sources were subjected to plasmid profile and ERIC-PCR characterization. We used a commercial plasmid extraction kit to extract and purify plasmid DNA which then was separated by gel electrophoresis and viewed under a UV transilluminator. For ERIC-PCR, a commercial bacterial chromosomal DNA extraction kit was used. In this study, we chose ERIC2 primer for the ERIC-PCR test. Results: The plasmid profile revealed that 35% of isolates did not contain any plasmids, but the rest (65%) carried a variable number of plasmids with different molecular weights. Six plasmid profiles were found among 40 S. Infantis isolates. Using ERIC2 primer, 7 profiles were found among 40 S. Infantis isolates in ERIC-PCR. Bands with molecular weights ranging from 400 to 3000 bp were observed. Conclusion: This study provided some genetic data on S. Infantis isolates recovered from poultry sources, and these data can be used for a broader epidemiological study nationwide. These findings showed that although plasmid and ERIC profiles are valuable in epidemiological studies, they have some limitations, too.

Abstract 1672: High-Quality Plasmid DNA Purification in 9 minutes following innovative FastFilter Technology

Abstract 1672: High-Quality Plasmid DNA Purification in 9 minutes following innovative FastFilter Technology

Rapid method for plasmid DNA recombination (Murakami-system)

DNA recombination is a useful technology for cloning and subsequent functional analysis, while standard techniques for plasmid DNA recombination have remained unchanged. In the present study, we introduced rapid method for plasmid DNA recombination, which we named “Murakami-system”, to complete the experiments in under 33 h. For this purpose, we selected the following: PCR amplification with 25 cycles and E. coli strain with rapid growth (incubation time of 6–8 h). In addition, we selected rapid plasmid DNA purification (mini-prep; ∼10 min) and rapid restriction enzyme incubation (20 min). This recombination system enabled rapid plasmid DNA recombination within 24–33 h, which could be useful in various fields. We also established a 1-day method for competent cell preparation. Our rapid recombination system allowed several sessions of plasmid DNA recombination to be performed every week, which improves the functional analysis of various genes.•“Rapid method for plasmid DNA recombination (Murakami-system).•E. coli strain with rapid growth (incubation time of 6–8 h).•Combination of rapid protocols (PCR, electrophoresis, DNA purification, ligation, and mini-prep) enabled plasmid DNA recombination within 24–33 h.

Secreting recombinant barnase by Lactococcus lactis and its application in reducing RNA from forages

Barnase is a ribonuclease used for plasmid purification, targeted gene therapy and studies of protein interactions. To make the use of barnase easier, the barnase gene from Bacillus amyloliquefaciens BH072 was cloned into Lactococcus lactis under the control of the PP5 or PnisA promoters. Four recombinant expression vectors were constructed with one or two signal peptides to control the enzyme secretion. 310 mg/L barnase was obtained in the presence of its inhibitor barstar after 36 h induction. The properties of barnase were investigated, showing that the optimal reaction temperature and pH were 50 °C and 5.0, respectively, and the highest enzyme activity reached 16.5 kU/mL. Barnase stored at 40 °C for 72 h retained 90 % of its initial activity, and maintained more than 80 % of its initial activity after 72 h of storage at pH 5.0–9.0. Furthermore, the optimal conditions for enzymatic reduction of nucleic acids in single-cell proteins (SCP) forages was investigated. 1 % salt solution with an SCP-enzyme ratio of 1000:1, pH 5.0 and incubated at 50 °C for 1 h, allowed 82 % RNA content reduction. Finally, homology modeling of barnase demonstrates its three-dimensional structure, and substrate simulation docking predicts key active residues as well as bonding patterns.

Homemade plasmid Miniprep solutions for affordable research in low-fund laboratories

As a consequence of Covid-19 pandemic, the basic lab consumables are in shortage, especially in the low-income countries. Thus, the main objective of the present study is to develop and evaluate homemade solution to isolate plasmid. To pursue this objective, RNase A was overexpressed in Bl21 DE3 cells (E. coli strain) and prepared as crude refolding reaction with proper activity. Also, lysis buffers, neutralization buffer, and washing buffers were prepared. The homemade miniprep kit showed successful isolation of the px48SpCas9 plasmid. The prepared plasmid purity was enough to be used successfully in PCR amplification. In addition, to get extra benefits from this study, seven primers were designed to match the plasmid backbone to produce DNA ladder (100–1500 bp). In conclusion, we were able to have attainable working solutions for plasmid miniprep and DNA ladder.