quantification
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what is the most important prepratory step in DNA profiling?
how much DNA has been obtained from an extraction
why is quantification important?
• estimate efficiency of extraction methods • for diluting samples to known concentration for use in PCR • for best PCR results in relatively narrow range of DNA
why should you dilute DNA samples?
conserving DNA, reducing the possibility of introducing inhibitors which may be present in the extracted solution
why is hair not a good idea for DNA quantification?
there is not a lot of DNA and there are a lot of inhibitors in the hair.
what copurifies when blood is soaked into dirt?
human acids
what does ethidium bromide do in gels?
when running uncut DNA through gel will allow you to estimate the size range and quality of DNA
how is good quality determined by weight?
it will be higher in weight.
what will degraded DNA do in the gel?
it will smear extending from 100-2000bp
what does ethidium bromide do for DNA?
makes DNA glow intercalating between double stranded DNA making it flouresce.
T/F: ethidium bromide only works for dsDNA.
true
what is better than ethidium bromide and why?
sybr green
what does multiple sizes on a smear mean?
it is cutting outside the histone
if DNA stays above ___ then it is high quality DNA.
2000bp
what is the maximal absorption for DNA and RNA?
260nm
what kind of relationship exists between the absorption of light and nucleic acid?
linear
what can A260/280 not do?
differentiate DNA to RNA
the ratio of A260/A280 indicates what?
contamination by protein or phenol
A ratio greater than 1.9 means what?
pure DNA
A ratio greater than 1.8 means what?
pure RNA
what is a ratio indicating lots of protein?
0.6
what is fluorescence?
absorbance of light at one wavelength and emission at a different wavelength
what does picogreen bind to?
preferentially to dsDNA
following contact with wavelengths of light what causes the picogreen to flouresce?
the amount of bound picogreen
stronger picogreen emissions implies what?
more DNA