281_DNA quantification and it's importance
- 2. Why? ▶ Assess quality DNA/RNA ▶ Determine amounts needed for downstream applications (Sequencing, PCR, Cloning) ▶ Confirm experiment / extraction success
- 3. Methods ▶ UV Spectrophotometry ▶Nanodrop ▶ Ethidium Bromide Staining ▶ Gel Electrophoresis Analysis ▶ Fluorometric Quantification ▶ PicoGreen ▶Qubit ▶Hoechst 33258 dye ▶ Real time PCR ▶ Bioanalyzer
- 4. UV Spectrophotometry ▶ Biomolecules absorb light in UV range. ▶ Allows us to estimate amount of DNA by its absorbance ▶ DNA: 260nm and 280nm ▶ Proteins: between 215-230nm and 280nm ▶ *Both proteins and DNA absorb light at 280nm. If sample is mixed, this can interfere with one another
- 5. UV Spectrophotometry: Nanodrop Disadvantages: ▶ Not species specific ▶ Bad resolution for low concentration samples (lower limit of 2ng/ul) ▶ Does not distinguish between ds or ssDNA ▶ Contaminating samples leads to falsely high quantitation readings Advantages: ▶ Uses small microvolumes (1-2 μl) ▶ Rapid results for quick assessments ▶ Graph gives indication of quality ▶ Widely used
- 6. UV Spectrophotometry: Nanodrop ▶ Absorbance of solution at two wavelengths ( 260nm and 280nm) ▶ Calculate ratio A260/A280 ▶ Ratio of less than 1.8 signifies that sample is contaminated with protein or phenol. Indications poor extraction. ▶ *dependent on pH and ionic strength of buffer Pure RNA: 2.0 Pure DNA: 1.8 Prue Protein: 0.6 Nucleic Acid Purity determined by: ▶ Absorbance of solution at two wavelengths ( 260nm and 230nm) ▶ Calculate ratio A260/A230 ▶ If ratio varies, may indicate presence of residual phenol, magnetic beads, carbohydrates. Pure RNA/DNA: 2.2-1.8
- 7. UV Spectrophotometry: Nanodrop Negative values: dirty pedestals or incorrect blank 1 2 Ragged line: Bad blank Jagged line: Broken read or low volume 3 4 High 230nm: Contaminates; carbohydrates, phenols, guanidine isothiocyanate
- 8. Other Methods ▶ Ethidium Bromide Staining: Binds to Nucleic Acid and gives orange fluorescence. ▶ Gel Electrophoresis Analysis; ▶ Calculate band size using software from imager. Compare fluorescence intensities of ladder and sample to estimate DNA concentration ▶ Create graph with linear trendline to calculate mass wrt intensity numbers Advantages: ▶ Specific bands ▶ Not pure samples Disadvantages: ▶ Need lots of DNA ▶ Not very accurate
- 9. Other Methods ▶ Fluorometric Quantification; uses fluorescent dye ▶ PicoGreen; binds dsDNA ▶ Measure fluorescent intensity of PicoGreen dye with spec. ▶ DNA quantified by comparing sample to set of standards ▶ Qubit; binds DNA, RNA or protein depending on kit ▶ Similar Advantages and Disadvantages to PicoGreen ▶ Hoechst 33258 dye; specific to DNA ▶ Good for both large and small amounts of DNA Disadvantages: Need special equipment and reagents/kit Longer prep time Advantages: High throughput Increased sensitivity Less prone to contaminants
- 10. Other Methods ▶ Real time PCR ▶ Fluorescent dye binding to dsDNA as it accumulates during PCR process ▶ Targets specific region of DNA template ▶ Used for sequencing prep, to verify quality and quantity of DNA libraries ▶ Bioanalyzer; automated electrophoresis ▶ Size, quantitation and purity assessments ▶ Small volume of sample needed ▶ Multiple platforms (RNA, DNA) ▶ Both low and high concentration samples ▶ Easy to use, but expensive ▶ Hybridization-Based Techniques; Southern or Northern Blotting ▶ “Probe sequence” based ▶ Higher resolution (down to actual nucleotide sequence)