Yield Gel via Quantitative Gel Electrophoresis.

Quantitative gel electrophoresis, also referred to as yield gel via gel electrophoresis, is an early quantification method that was developed to provide an estimate of the quality and the quantity of DNA extracted from evidence or reference samples. To conduct quantitative gel electrophoresis, an agarose gel that is combined with a nucleic acid gel stain is prepared. The gel stain intercalates between double-stranded DNA and can be visualized using UV light. DNA extract samples, along with DNA standards (ranging from 250 to 5 ng), and a 1 KB ladder are combined with a 6X loading dye and loaded on the agarose gel. Voltage is applied to facilitate DNA migration through the gel from the negative to the positive electrode, separating DNA fragments by size. After electrophoresis is complete, the results are visualized using UV light, and an image is captured for analysis. High-quality and -quantity DNA should contain a compact band comparable to that of the high molecular weight standards and ladder, with some smearing down the sample well. If a DNA extract sample does not produce a compact band and presents with only a smear, this is an indication that DNA degradation has occurred. This chapter provides instructions on how to successfully prepare an agarose gel, load DNA extract samples and corresponding controls, appropriately set up and run quantitative gel electrophoresis, interpret the results, and ensure comprehension of the method so troubleshooting can be performed if needed.

Quantitative PCR of Alu Repeats Using PowerUp™ SYBR® Green Master Mix.

Quantitative PCR is one of the fundamental steps performed when processing routine casework in a forensic laboratory. Quantitative PCR of Alu repeats using a SYBR® Green master mix can produce calculated estimates of how much DNA was extracted from a sample. This process offers more efficiency, human specificity, and can be performed faster than other outdated quantification methods, such as slot blot or yield gel. A qPCR master mix is prepared and consists of Alu-F primers, Alu-R primers, water, and SYBR® Green master mix. The Alu-F and Alu-R primers target Alu sequences that are present hundreds of thousands of times throughout the human genome and are effective markers for human DNA quantification. During qPCR, the 7500 system facilitates the amplification of target Alu repeats. The SYBR® Green I fluorescent dye intercalates between the amplified dsDNA targets. During each amplification cycle, the 7500 system agitates the SYBR® Green I dye, resulting in a fluorescence signal that is recorded when it passes a specified Ct value. After qPCR amplification is complete, a standard curve is created and used to determine how much DNA a sample contains. This chapter provides instructions on how to accurately prepare a 96-well plate for qPCR, use the 7500 system and associated software to set up the qPCR amplification, and interpret the corresponding results produced.