Alkaline Agarose Gel Electrophoresis

Alkaline agarose gels are run at high pH, which causes each thymine and guanine residue to lose a proton and thus prevents the formation of hydrogen bonds with their adenine and cytosine partners. The denatured DNA is maintained in a single-stranded state and migrates through an alkaline agarose gel as a function of its size. Other denaturants such as formamide and urea do not work well because they cause the agarose to become rubbery.

Identification of Polymorphisms of the CSN2 Gene Encoding β-Casein in Greek Local Breeds of Cattle

This research focused on the detection and identification of genetic polymorphisms in exon 7 of the β-casein CSN2 gene in blood samples from Greek Holstein cows and from local breeds of cattle, such as Vrachykeratiki, Katerinis, and Sykias. For this purpose, DNA was isolated from 780 blood samples obtained from Greek Holstein cows, 86 from three local breeds of cattle, namely Brachyceros, Katerinis, and Sykias, and 14 from Greek buffalo. The desired region of exon 7 was amplified by PCR, resulting in 121 and 251 bp products in bovine and buffalo samples. The PCR product was digested with restriction fragment length polymorphism (RFLP) on agarose gels. The restriction enzymes DdeI and TaqI were used. All of the blood samples had the amplified size. The results showed that 74.4% of the Greek Holstein cows had the A2A2 β-casein genotype, the three native breads Vrachykeratiki had 57.7%, and the other two had 100% of the A2A2 β-casein. From the 14 Greek buffalo ,100% had the A2A2 β-casein.

Automatic Lab on PCB System for Agarose Gel Preparation and Electrophoresis for Biomedical Applications

In this paper, a prototype of an automatic lab on PCB for agarose preparation and electrophoresis is developed. The dimensions of the device are 38×34 mm2 and includes a conductivity sensor for detecting the TAE buffer (Tris-Acetate-EDTA buffer), a microheater for mixing, a NTC thermistor for controlling the temperature, a LDR sensor for measuring the transparency of the mixture, and two electrodes for performing the electrophoresis. The agarose preparation functions are governed by a microcontroller. The device requires a PMMA structure to define the wells of the agarose gel, and to release the electrodes from the agarose. The maximum voltage and current that the system requires are 40 V to perform the electrophoresis, and 1 A for activating the microheater. The chosen temperature for mixing is 80ºC, with a mixing time of 10 min. In addition, the curing time is about 30 min. This device is intended to be integrated as a part of a larger lab on PCB system for DNA amplification and detection. However, it can be used to migrate DNA amplified in conventional thermocyclers. Moreover, the device can be modified for preparing larger agarose gels and performing electrophoresis in an automatic manner.

Using Fluorescence in Biotechnology Instruction to Visualize Antibiotic Resistance & DNA

Fluorescence technology has many useful applications for both research and teaching, among them the detection of fluorescence in live transgenic organisms and of DNA in agarose gels. However, dedicated fluorescence imaging systems can be expensive and complex. We describe a simple apparatus for non-microscopic fluorescence imaging using affordable and readily available parts. We describe three activities of increasing complexity that utilize fluorescence illumination and teach principles of fluorescence. At the high school level, our more advanced activities can be used for lessons addressing NGSS performance expectations in HS-LS3 and HS-LS4.

Plasmid supercoiling decreases during the dark phase in cyanobacteria: a clarification of the interpretation of chloroquine-agarose gels.

In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with the circadian transcription program and (Woelfle et al. [2007], Vijayan et al. [2009], PNAS). Specifically, Woelfle et al. have reported that DNA supercoiling of an endogenous plasmid became progressively higher during prolonged dark phases in Synechococcus elongatus PCC 7942. This is counterintuitive, since higher levels of negative DNA supercoiling are commonly associated with exponential growth and high metabolic flux. Vijayan et al. then have reverted the interpretation of plasmid mobility on agarose gels supplemented with chloroquine diphosphate (CQ), but not further discussed the differences. Here, we set out to clarify this open issue in cyanobacterial DNA supercoiling dynamics. We first re-capitulate Keller's band counting method (1975, PNAS) using CQ instead of ethidium bromide as the intercalating agent. A 500x-1000x higher CQ concentration is required in the DNA relaxation reaction (topoisomerase I) than in the agarose gel buffer to quench all negative supercoiling of pUC19 extracted from Escherichia coli. This is likely due to the dependence of both, the DNA binding affinity of CQ and the induced DNA unwinding angle, on the ionic strength of the buffer. Lower levels of CQ were required to fully relax in vivo pUC19 supercoiling than were used by Woelfle et al. Next, we analyzed the in vivo supercoiling of endogenous plasmids of Synechocystis sp. PCC 6803, at two different CQ concentrations. This clearly indicates that negative supercoiling of plasmids does not increase but decreases in the dark phase, and progressively decreases further in prolonged darkness.

Tuning Transport Phenomena in Agarose Gels for the Control of Protein Nucleation Density and Crystal Form

Agarose gels provide the ideal environment for studying the nucleation step of complex biomacromolecules under diffusion-controlled conditions. In the present paper, we characterized the influence of agarose on the nucleation of three model proteins, i.e., lysozyme, insulin, and proteinase K, as a function of the agarose concentration using a batch method set-up inside flat capillaries. By using this set-up, we were able to directly count the number of crystals in a given volume and correlate it with the amount of agarose and with the average crystal size. We also studied the crystallization behavior of proteinase K with free-interface diffusion so that batch conditions were achieved through slow diffusion of the precipitant. Thanks to the control over the protein mass transport imposed by the network, a previously unknown crystal form, P212121, was obtained, and the three-dimensional structure was determined at a 1.6 Å resolution. Overall, the versatility of agarose gels makes them ideal candidates for the preparation of microcrystalline suspensions of biopharmaceuticals with precise and reproducible crystal attributes or for the exploration of the existence of different polymorphs.

An Electrochemical System for Forming Periodic Precipitation Bands of Cu-Fe-Based Prussian Blue Analogues

We propose a novel electrochemical system to form precipitation patterns of Cu-Fe-based Prussian blue analogues (Cu-Fe PBA) in agarose gels, using an applied voltage to produce reactant ions. The spatiotemporal evolution, spatial distribution, and crystallite morphologies of the precipitates were investigated by visual inspection, Fe Kα intensity distribution measurements, and optical and scanning electron microscope observations. The precipitation patterns and their evolution depended on the applied voltage. Multicolored periodic precipitation bands were stochastically formed under cyclic alternating voltage (4 V for 1 h and then 1 V for 4 h per cycle). The distances between adjacent bands were randomly distributed (0.30 ± 0.25 mm). The sizes and shapes of the crystallites generated in the gel were position-dependent. Almost cubic but fairly irregular crystallites (0.1–0.8 μm) were formed in the periodic bands, whereas definitely cube-shaped crystallites (1–3 μm) appeared close to the anode. These cube-like reddish-brown crystallites were assigned to Cu-FeII PBA. In some periodic bands, plate-like blue crystallites (assigned to Cu(OH)2) were also present. Future issues for applications of the observed periodic banding were discussed.