Case Study: Agarose Gel Electrophoresis

3.8.2. Agarose Gel Electrophoresis
Performed according to Brody et al.
Reagents
1. Agarose
2. Tris borate EDTA (TBE) buffer, pH-8.0,1x buffer solution.
3. Ethidium bromide (EtBr).
4. Bromophenol blue dye- Low molecular weight dye for tracking the progress of electrophoresis.
5. DNA sample.
Procedure
2 g agarose was weighed in a conical flask, mixed with TBE buffer (100 ml ) and boiled in a microwave oven to dissolve the agarose and obtain a clear solution. The solution was cooled to a lower temperature and mixed with 2µl of EtBr. This solution was poured into a trough which contained a comb, for polymerization. Then this stacked gel was placed in the tank which contained TBE buffer and then the comb was removed carefully for the formation

20ul
2. Turn on PCR machine.
3. Input sample identifiers on qPCR running software, including NTC, positive control and quantities of standards.
4. Cycling conditions, followed by a dissociation (or melt) curve are as below
REACTION CONDITIONS TIME IN Sec.
95°C 30
95°C 5 40 CYCLES
60°C

5. Processing and analyzing the data
The values are then exported (kb/reaction for telomere and genome copies/reaction for SCG) to csv format. The kb/reaction value is then used to calculate total telomere length in kb per rat diploid genome. The telomere length in kb per reaction value is divided by diploid genome copy number (2n=42) to give a total telomeric length in kb per rat diploid genome.
3.9. Evaluation of alterations in the neurochemical profile
NMR spectroscopy is the most important tool in the study of metabolic processes and fluxes, involved in brain energy metabolism, non-invasively in vivo. NMR technologies have become more helpful, providing the quantitative assessment of transport steps, metabolic fluxes and cellular compartmentalization of glycolysis, pyruvate oxidation, and tricarboxylic acid (TCA) cycle and other metabolic pathways. 13C NMR approaches constitute probably the most elaborated, chemically specific, tool to follow the metabolic fate of 13C labeled substrates in the brain, both in vivo and in vitro.