UV adsorbtion

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Mol.biology // Next-generation sequencing

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[править] Protocol

  1. dilute NA in 0.7-1x STE (mix thoroughly and spin down);
  2. prepare the same dilution of H2O in STE as a control;
  3. measure control as a blank;
  4. measure a sample



[править] Notes

  • A(Absorbance) = -log (Intensitysample/Intensityblank). The highest accuracy reached is in the range 0.3 < A260 < 0.7. It is impossible to measure accurately "high" and "low" absorbances because:
» high Absorbance means, that "Intensitysample" is very low. It is difficult for spectrophotometer to measure a low signal.
» low Absorbance means, that "Intensitysample" is practically the same as the "Intensityblank". It is difficult for spectrophotometer to distinguish similar signals.
  • adsorbtion coefficients for NA in 1x STE (0.1M NaCl, 10mM Tris-Cl, 1mM EDTA, pH7.5):
c[µg/ml] = k * A260
NAk [µg/ml]
dsDNA50
ssDNA37
ssRNA40
oligovariable
  • 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. For dsDNA k [µg/ml]:
» in H2O: 38.1
» in TE: 44.9
» in TE + saline: 50
  • it is important to use as a "control" as much as possible the same solution as for the sample. For example, if "sample" is {90µl of 1x STE & 10µl of DNA}, then "control" should be {90µl of 1x STE & 10µl of H2O};
  • UV adsorbtion is the only accurate method of quantitation of oligonucleotides. Adsorbtion coefficient for oligos is sequence-dependent. Online analysis: Oligo Analyzer 3.1
  • chemical or enzimatic digested of NA does not decrease UV-adsorbance significantly. If the sample containes "small RNA", "degraded RNA", "degraded DNA" use either "in gel" or fluorescent quantification.
  • it is important to control the shape of the adsorbtion spectrum:
▫ to distinguish small signals from noise
▫ to take "medium level of " A320-A340 as a "zero-level"
  • it is difficult to use shape of UV-spectrum as a measure of Nucleic acid purity (description is under the "+")


[править] Spectrophotometer

  • normally, 100µl cuvette is used. Mix samples in cuvette 2-3 times by pipetting.
  • "zero-level" should be taken in A320-A340 range.
  • accuracy:
A260accuracy
~0.005~20%
~0.01~10%
0.3-0.7~0.3%
0.1-0.3 and 0.7-1~1%
>2.5unreliable
  • use water pump for aspiration of liquid from the cuvette.
  • newer use air stream for blowing out drops from the cuvette. It leads to DNA contamination of laboratory.
  • cuvette washing:
▫ no need to wash between mesurements of samples with comparable concentrations: aspirate the old sample (with a pump), load the next;
▫ routine wash (at the beginning, in the end of the work and between samples with unknown concentration): 2-3 times with bidistilled water (pour water and aspirate it by a vacuum pump);
▫ extremely clean wash (if sample would be used in an experiment after measurement):
▪ 2 h in methanol:HCl=1:1
▪ rinse several times in mQ H2O.



[править] Nanodrop

  • specification:
sample size1-2µl
path length1mm (with auto-ranging to 0.2mm)
minimal amount~2-10ng
maximum amount~3µg
highest accuracy0.2-0.5µg
detector type2048-element linear silicon CCD array
wavelength range220-750nm
absorbance precision0.003 absorbance (1mm path)
measurement time10 seconds
  • Nanodrop automatically takes A340 as a zero-level.
  • it is important to control that liquid bridge was formed:
▫ 1µl is enough for buffers with a high surface tension (H2O, TE, EB, STE)
▫ 2µl - for buffers with low surface tension (detergents, proteins)
  • remove solutions without scratching the surfaces of the pedestals (blot with a paper towel or aspirate with a water pump)!


[править] Washing

  • washing between samples:
▫ usually, it is enough to remove the previous sample from upper and lower pedestals;
▫ if necessary, clean surfaces with ~5µl of dH20;
  • regular washing:
○○   repeat twice:
▫ apply ~5µl of dH20 solution onto bottom pedestal
▪ water pump for aspiration (better)
▪ paper towel bloting (worth)
▫ lower the upper pedestal to form a liquid column; leave it for approximately 2-3 minutes
▫ remove water from upper and lower pedestals
  • special washing:
» decontamination: 0.5% solution of sodium hypochlorite (1:10 dilution of common commercial bleach solutions – freshly prepared)
» reconditioning: rubbing each measurement surface aggressively with a dry laboratory wipe 30-40 times. Buffers containing surfactants, the Bradford reagent can "un-condition" the measurement pedestal surfaces so that the liquid column does not form well with 1µl samples.



[править] Comparison of Spectrophotometer and Nanodrop

  • S>N: more stable measurements, ~5% variations between independent measurements for spectrophotometer, and ~30% for Nanodrop
  • N>S: ~10 times higher sensitivity for Nanodrop. Detection limit for spectrophotometer is ~100ng, for Nanodrop: ~2-10ng
  • N>S: handling


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