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DNA 3 Controls

Controls and Quantitation

How many diploid cells are in 1n of human DNA? 1 cell contains 6 pg of DNA. 1000pg - 6 ng.??????????
Controls Must know the quality and quantity of the control before use. Quantify with sensitive assay, Qualify with gel electrophoresis. Make serials dilutions from 10ng to 10pg Make aliquots to avoid contamination and degradation
Methods of DNA Quantitation? UV 280/260 Not human specfic, not very sensitive or DNA specific. Yield Gel: Not human Specific, tell quality,can guess quantity, not sensitive Fluorescence:Not Human Specific, more sensitive Slot Blot:Human Specific, sensitive, poor dynamic range.
Other Method of DNA Quantitation? RT qPCR: Human Specific, very sensitive, good dynamic range.
What is the Slot Blot Method? Method allows is simple and allows you to determine the concentration of an unknown sample of DNA relative to a standard. Samples blotted onto a membrane(max 30)along with serially diluted standard of known concentration. Visual comparison to standard
What is the Slot Blot Method? More Visualized using a probe that is specific to a region in chromosome 17. Human or primate specific. Can detect single and double stranded DNA as low as 150pg. (50 copies of human DNA) Can get as low as 10-20 pg of DNA. Gel results have limited comparis
Explain Real Time Quantitative PCR RT qPCR Quantifies DNA as it is amplfied. Utillizes SYRB Green or Flouresent probes for detection. Allows you to quantify the amount of amplifiable DNA.
Slot Blot versus 2-3 hours of rinses, incubation, pipeting, wash, exposures, developments Involves comparison to standards run simultaneously Semi quantitative by manual comparison or through scanner Quantity obtained will not reflect PCR inhibitors. Real Time qPCR 1 hour set up 2 hour run time Comparison to standards run simultaneously Automated quanitiation Quality obtained reflects amount of implied DNA
END Point PCR Less elegant than RT qPCR, but less expensive Single STR locus or region is amplified with DNA samples of known concentration Tells you Quality of amplifiable DNA DYE such as SYBR Green is used to detect PCR products on a gel. Quantitation=intensity.
Alu Quant It is a restriction enzyme region. Alu repeats are common in primates and people. Human specific Range=0.1 to 50 ng Automated Probe modified ending with a luciferin molecule. Results in emission of light.
Nano drop 0.5 to 2 ul samples is needed. Sample is pipeted directly onto the pedestal Then it automatically adjusts to optimal math length
Pico Green Assay Automated, high throughput method for rapid DNA quantitation Can detect as little as 0.25nm/ml of double stranded DNA in a 95 well plate.
Created by: Perrin Serroul Perrin Serroul