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Spectrophotometric quantitation

Spectrophotometer Nanodrop Qubit Nano-fluorometer  Comments

UV adsorbtion (spectrophotometer & Nanodrop)

• adsorbtion coefficients (c[µg/ml]=k*A₂₆₀) for NA in 1x STE (0.1M NaCl, 10mM Tris-Cl, 1mM EDTA, pH7.5):

  NA k [µg/ml] dsDNA 50 ssDNA 37 ssRNA 40 oligo variable

• these coefficients are valid for the measurements in buffered medium-salt solutions (pH7-8; 20-100mM salt). Mesurement in water results in overestimation of NA concentration:

  solution k [µg/ml] H2O 38.1 TE 44.9 TE + saline 50

• it is important to use as a "control" as much as possible the same solution as for the sample. It means, that if "sample" is {90µl of 1x STE & 10µl of DNA}, then "control" should be {90µl of 1x STE & 10µl of H₂O};
• the highest accuracy reached is in the range 0.3<A₂₆₀<0.7;
• it is important to control the shape of the adsorbtion spectrum:
  • to distinguish small signals from noise;
  • to take A₃₂₀-A₃₄₀ is a "zero-level";
  • some buffers have strong adsorbance in UV-range and interfere with measurement;
• A₂₆₀/A₂₈₀ ratio is unreliable parameter for control of RNA contamination. It has completely no sense if NA is not in the buffered solution such as Tris-HCl, KHPO₄, 10-100mM, pH7-8;
• NB! chemically or enzimatically digested NA have ~same UV-adsorbance. If the sample containes {small RNA}, {degradated RNA}, {degradated DNA} use either "in gel" quantification or fluorescent quantification.

Spectrophotometer

• program for Spectrophotometric Measurement of Nucleic Acids' Concentration;
• NB! A₃₂₀ should be taken as "zero-level";
• accuracy:

  A260 accuracy ~0.005 ~20% ~0.01 ~10% 0.3-0.7 ~0.3% 0.1-1.0 ~1.0% >2.5 unreliable

Maintenance

• cuvette washing:
  • between samples with comparable concentration: aspirate the old sample, do not wash cuvette;
  • normal (at the beginning, in the end of the work and between samples): 1÷3 times with bidist-water (pour wather and aspirate it by wacuum pump);
  • extremely clean (sample will be used in the experiment after measurement): (i) 2 h in methanol:HCl=1:1, rinze in mQ H₂O;



• use water pump for aspiration of liquid from the cuvette;

Nanodrop

• it is important to control that liquid bridge was formed;
• 1µl is enough for buffers with a high surface tension (H₂O, TE, EB, STE); 2µ - with low (detergents, proteins);
• Nanodrop automatically takes A₃₄₀ as a zero-level;

Maintenance

• wipe away solutions without scratching!



• regular washing, repeat twice:
  1. apply ~5µl of dH₂0 solution onto bottom pedestal;
  2. lower the upper pedestal to form a liquid column; let it sit for approximately 2-3 minutes;
  3. wipe away the water form each upper and lower pedestal with a paper towel (or water pump);



• washing between samples:
  • normally, it is enough to wipe away the previous sample from upper and lower pedestals with a paper towel (or water pump);
  • if necessary, clen surfaces with ~5µl of dH₂0;

UV adsorbtion (spectrophotometer & Nanodrop)

Qubit

Maintenance

Nano-fluorometer

Maintenance