Controls and Quantitation
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| How many diploid cells are in 1n of human DNA? | 1 cell contains 6 pg of DNA. 1000pg - 6 ng.??????????
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| 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
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| 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.
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| Other Method of DNA Quantitation? | RT qPCR: Human Specific, very sensitive, good dynamic range.
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| 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
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| 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
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| 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.
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| 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
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| 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.
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| 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.
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| 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
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| 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.
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