Transformation and isolation

The transformation and isolation of the pCAMBIA 1301 plasmid was carried out as follows: In the PCR reaction, pCAMBIA 1301 plasmid was used as a template DNA. The plasmid was then transformed into Escherichia coli (Top 10) bacteria using the heat shock method as described by [9]. The transformed Escherichia coli (Top 10) bacteria containing our desired plasmid (pCAMBIA 1301) were cultured on LB agar (Luria-Bertani medium) plates containing Kanamycin antibiotic (50 µg/ml). A single colony of bacteria (transformants) was cultured again in LB broth containing Kanamycin antibiotic overnight. The next day, the pCAMBIA 1301 plasmid was purified using a kit method (Thermo Scientific GeneJET Plasmid Miniprep Kit #K0502, #K0503), and this plasmid was used as a template DNA in subsequent PCR reactions.

Designing of PCR primers. According to the plasmid DNA sequence (pCAMBIA 1301 plasmid), 10 pairs of primers were designed to amplify 100-1000 base pair fragments using the online tool Primer3Plus. These primers were purchased from Macrogen Company (Korea) for the successful amplification of the expected DNA fragments ranging from 100 to 1000 base pairs. The sequences and temperature of each primer are shown in Table 1, where "F" shows the forward strand, while "R" shows the reverse strand of the primer.

Optimization of fragments 100 base pair to 1000 base pair using gradient thermocycler

For each DNA fragment, a separate PCR reaction was performed in a 20 µl volume, containing 1.5 µl of the pCAMBIA plasmid template DNA, 2 µl of dNTP (2mM), 2 µl of Taq buffer (10x), 1.5 µl of MgCl2, 9.5 µl of PCR water (Thermo Fisher Scientific, USA), 3 µl of both forward and reverse primers (10 pmol each), and 0.5 µl of Taq DNA polymerase (5U/ µl, Thermo Fisher Scientific, USA). To amplify all DNA fragments at once, the optimal annealing temperature was determined using a temperature gradient ranging from 57-61°C. The PCR cycle program was designed in a thermal cycler with an initial denaturation step at 95°C for 6 min, followed by 36 cycles of denaturation at 95°C for 35 sec, annealing at 59-61°C for 35 sec (as illustrated in Table 1), and extension at 72°C for 35 sec. After the 36 cycles, a final extension was carried out at 72°C for 7 minutes.

PCR product purification

The PCR products of 100 to 1000 base pair were purified using the purification kit method (WizPrep^TM Gel/PCR Purification Mini Kit) as instructed. Briefly, PCR products were treated with a PCR purification buffer (PCR purification buffer: PCR sample = 5:1). The mixture was transferred to the spin column and centrifuged at 14000 rpm for 1 minute. The flow-through after centrifuge was discarded and column re-inserted into the collection tube and samples were washed by 700 µl wash buffer (ethanol added) centrifuged for 30 sec. The supernatant was again discarded, and the spin column was centrifuged for 1 minute at 14000 rpm for the removal of residual Wash buffer completely. Spin column was placed in an autoclaved 1.5 ml tube and 40 µl elution buffer was added to the center of the spin column and placed for 1 to 2 minutes at room temperature and centrifuged at 14000 rpm for 1 minute for DNA elution.

Identification and sequencing of DNA fragments

A total of 4 µl (150 ng) of each PCR-purified product was sequenced on a 2% agarose gel stained with Ethidium Bromide (Thermo Fisher Scientific, USA). The products were then electrophoresed in 1X TBE buffer (Tris base, boric acid, EDTA) at 80 volts for 120 minutes, and the length of fragments was estimated by comparing to a 100 bp DNA size marker (Biolis Biodyne, 100 bp). The appearance of DNA bands was visualized under UV light at 260/280 nm on a gel documentation system. Purified DNA fragments were sequenced using the facilities of 1st Base Sequencing, Malaysia. The received sequences were analyzed using the Bio-Edit tool to compare the length and number of base pairs of fragments with the template DNA.

Stability and evaluation of 100base pair DNA ladder

Various sizes of purified PCR products were mixed according to the specific proportions and dissolved in seven different buffers (shown in Table 2) for DNA ladder stability. The ladder was ready for laboratory use and was compared with the commercial version. The 100 base pair DNA ladder was stored at -20°C. Finally, the cost-effectiveness of the 100 base pair DNA ladder was estimated using the Break-Even Point formula in Microsoft Excel.  

Figures & Tables

The scientific examinations depend on standard units for the measurement of quantitative descriptions of natural phenomena. In molecular biology, scientists have established standard units for genomics to develop a DNA ladder that expresses the quantitative standard unit for the measurement of unknown DNA fragments by acting as a scale to measure genetic features like human genetic variation and microbial variation [10]. DNA ladder is essential and cost-effective in molecular biological research and is available commercially which is too expensive for laboratory usage. Therefore, the development of an indigenous DNA ladder can result in cost-effective strategies to curb expenses.  In this study the main objective was to prepare cost-effective 100 base pair DNA ladder prototype with fragments of 100-1000 base pair with 100 base pair intervals.

For the development of the prototype plant expression vector, pCAMBIA 1301 was used as a templet. About 10 pairs of primers were designed from the backbone of the vector, ranging from 100 base pairs up to 1000 base pairs, with an interval of 100 base pairs. Each fragment was amplified using thermocycler and resolved on gel electrophoresis. The outcome of PCR product on agarose gel showed that all of the 10 experimental fragments (ranging from 100 to 1000 base pair) were exactly aligned with the respective bands of DNA ladder in the standard column as shown in the results. The fragments were subsequently purified and quantified using a nanodrop, then sequenced using the facility of 1st base Sequencing, Malaysia. Each purified fragment was pooled with seven different buffers and stored at -20°C.The stability of the ladder containing different buffers was evaluated on an agarose gel every 15 days up to 180 days. Further, costing was done using break even analysis.

Wang et al., prepared a DNA ladder with 12 fragments ranging from 100 to 1500 base pairs. The study resulted in the amplification of DNA fragments by PCR. Hekmatnezhad, et al., [14] also produced DNA ladders using different types of forward and reverse primers. The results of [11] showed successful amplification of DNA fragments ranging from 100 to 1000 base pairs in a single PCR reaction for the production of DNA ladder for various research purposes in molecular biology experiments. According to the researchers, the PCR product mixture was confirmed by agarose gel electrophoresis and used as a DNA ladder without any further purification. These unpurified PCR products were as reliable as markers from commercial sources. Similarly, [4] amplified 11 different DNA fragments ranging from 100 to 1,000 base pair, as well as a 1,500 base pair fragment, using PCR. Finally, [12] successfully produced and tested a standard ladder through PCR. Producing a 100 base pair DNA ladder through PCR is a simple, inexpensive, and time-saving method for laboratories.

Another technique for producing DNA fragments of various lengths is performed through Restriction enzymes (endonucleases). Henrici et al., conducted a study on pPSU1 & pPSU2 pairs of plasmids to produce 100 base pair and 1 kb DNA ladders by the digestion of two common restriction enzymes [15]. These pair of plasmids are used as reference fragments from 50 to 1000 base pair at a fraction of the cost of commercial DNA ladders. Hartley also invented the nucleic acid marker ladder through restriction endonuclease digestion, using restriction enzymes digestion a collection of DNA fragments resulting from complete digestion of one or more DNA fragments by one or more restriction endonucleases.

In our study, the purified DNA product of the DNA ladder was treated with various DNA stabilizers. Solutions with different concentrations, as cited in the literature, were used as stabilizing media for the DNA fragments. The solution containing DNA fragments was mixed with the stabilizing media and then stored at -20 °C for the further testing procedure. The stability of DNA fragments was tested for 6 months post initial storage.

Wang et al. [11] studied DNA fragment intensities, finding that the intensity of each fragment was proportional to its size and length, resulting in a lower intensity for the 100 base pair fragment compared to larger fragments. The researchers mixed different concentrations of DNA fragments to construct the DNA ladder for visualization. Similarly, Baoutina et al. stored 20 DNA fragments in TE buffer and distilled water at different temperatures, studying them for 10 weeks. Over this time period, 6 of the 20 DNA fragments dissolved in TE buffer stored at 25°C and 4 of the 20 fragments dissolved in distilled water stored at 4°C were degraded. However, the 20 DNA samples stored in TE buffer at 4°C remained stable for 10 weeks. Additionally, the DNA samples preserved at -20°C and -70°C were not degraded in either distilled water or TE buffer, even after 10 weeks. According to the study in [13], TE buffer is a good stabilizer for DNA samples, and can be used to preserve and store DNA fragments at -20°C or -70°C for an extended period.

In research, scientists use DNA ladder in the laboratory regularly for different purposes. However, commercial DNA ladders can be expensive and can affect the financial cost of researchers. Therefore, we developed a cost-effective prototype of a 100 base pair DNA ladder or marker using the polymerase chain reaction technique. We stored the prototype DNA ladder in different buffers to check its stability. Using our methodology, researchers can easily prepare a 100 base pair DNA ladder in the laboratory within a few days. This strategy is an effective way to reduce costs and improve the availability of DNA ladders for laboratories.

Author Contributions

All of the authors contributed in conception, acquisition of data analysis, design, drafting, interpretation of data and revising the article.

Conflict of Interest

The authors declare that there is no conflict of interest.

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