M. Fero 11/05
Adapted from Turner's protocol (PDF) (website)
DNA: Höchst 33258 (High range solution): (Invitrogen H1398 or Sigma B1155) 1µg/mL in 1x TNE
Filter and store at 4ºC in an amber bottle.
10x TNE stock: 100 mM Tris, 2 M NaCl, 10 mM EDTA, pH7.4. Store at room temp.
RNA: Ribogreen 200x (Invitrogen R-11491) detection reagent. Store dessicated at -20ºC.
Turner Designs TD-360 Fluorometer
TE pH 8 (DNA), pH 7.5 (RNA)
Discussion
Fluorometry utilizes fluorescent dyes which specifically bind DNA or RNA. It requires a negative control (to set the zero point on the fluorometer) and a standard of known concentration. The fluorometer shines light on the sample (excitation) and then measues level of fluorescent light being emitted to the side (at a 90º angle) of the excitation light beam. The fluorescent dyes are relatively specific to nucleic acids as opposed to protein and other cellular components. The fluorescence of the dyes increases when they bind nucleic acids. Fluorometry is about 1,000x more sensitive than spectrophotometric absorbance (i.e. measurement of A260) and less susceptible to protein and RNA contamination. However it also does not give a crude measurement of purity (like an A260/A280 ratio) nor does it assure that the DNA or RNA is not degraded (e.g. like size determination by gel electrophoesis). Do not use glass (spectrophotmetry) cuvettes in a fluorometer because the frosted glass on the side of the cuvette interferes with detection of fluorescent light.
Detection Ranges
Procedure (High range assay solution)
Adapted from Turner's protocol (PDF) (website)
Fluorometric
Quantitation of RNA or DNA
MaterialsDNA: Höchst 33258 (High range solution): (Invitrogen H1398 or Sigma B1155) 1µg/mL in 1x TNE
Filter and store at 4ºC in an amber bottle.
10x TNE stock: 100 mM Tris, 2 M NaCl, 10 mM EDTA, pH7.4. Store at room temp.
RNA: Ribogreen 200x (Invitrogen R-11491) detection reagent. Store dessicated at -20ºC.
Turner Designs TD-360 Fluorometer
DNA: Long UV LED
(underneath) and filter set (inside chamber)
RNA: Blue LED and filter set
RNA or DNA standards (100 ng/µL)RNA: Blue LED and filter set
TE pH 8 (DNA), pH 7.5 (RNA)
Discussion
Fluorometry utilizes fluorescent dyes which specifically bind DNA or RNA. It requires a negative control (to set the zero point on the fluorometer) and a standard of known concentration. The fluorometer shines light on the sample (excitation) and then measues level of fluorescent light being emitted to the side (at a 90º angle) of the excitation light beam. The fluorescent dyes are relatively specific to nucleic acids as opposed to protein and other cellular components. The fluorescence of the dyes increases when they bind nucleic acids. Fluorometry is about 1,000x more sensitive than spectrophotometric absorbance (i.e. measurement of A260) and less susceptible to protein and RNA contamination. However it also does not give a crude measurement of purity (like an A260/A280 ratio) nor does it assure that the DNA or RNA is not degraded (e.g. like size determination by gel electrophoesis). Do not use glass (spectrophotmetry) cuvettes in a fluorometer because the frosted glass on the side of the cuvette interferes with detection of fluorescent light.
Detection Ranges
| Large
Cuvette |
Mini-cuvette |
|
| High Range Solution |
20 ng - 1 µg |
1 - 50 ng |
| Low Range Solution |
1 - 50 ng |
0.1 - 5 ng |
Procedure (High range assay solution)
- Insert the correct LED and filter set in the fluorometer for either RNA or DNA.
- Turn on the Fluorometer to warm up for 5 min.
- DNA: Remove Höchst (high range) solution from the
refrigerator. Dilute Höchst 1/10 (v/v) in 1x TNE if a low
range assay is being performed.
RNA: Thaw Ribogreen and dilute to 1/200 (high range) or 1/2000 (low
range) in
1x RNAse-free TE (pH 7.5). - Large cuvettes require 1.5 mL/sample
- Mini-cuvettes need 100 µL/sample.
- Aliquot 1.5 mL (large cuvette) or 0.1 mL
(microcuvette) of either Höchst or Ribogreen reagent into 1.5 mL
eppendorf tubes.
- Add 1.5 µL of DNA or RNA to each tube and vortex. In addition to samples this must also include a known standard and a TE or H2O control.
- Using narrow (gel loading) tips pipet the TE or H2O
control in a cuvette, close the chamber's lid and press the "Blank"
button. Press "1" when prompted to save the data. Open the
chamber's lid and transfer the control mix back to its original
Eppendorf tube.
- Transfer the DNA or RNA standard to the cuvette and press the "Calibrate" button. When prompted enter the (undiluted) concentration of the standard (i.e. 100) plus the "Enter" button. Press "1" when prompted to save the data.
- Repeat the zero and calibration steps (6 and
7). This is important to avoid negative values at low
concentrations.
- Measure each RNA sample and record the
concentrations.
Make up enough reagent for the samples plus a blank and at least one control.
