NA quantitation

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Mol.biology // Next-generation sequencing // Wet laboratory

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 UV adsorbtionintercalator fluorescencesize selection and interc. fluorescence
 Ultraspec 2100 proNanodrop 1000[1]QubitNanoDrop 3300 Fluorospectrometer[1]ElectrophoresisMultiNABioAnalyser
detection range0.1-7µg2ng - 3µgBR: 3ng - 1µg
HS: 0.2-100ng
RNA: 5-100ng
BR: 10pg - 5ng
HS: 1-500pg
EtBr: 10ng - 1µgDNA: 0.2-50ng
total RNA: 5-250ng
mRNA: 5–250ng
DNA: 0.2–50ng
RNA 6000 Nano
total: 5-500ng
mRNA: 25-250ng
RNA 6000 Pico
total: 50pg – 5ng
mRNA: 0.25–5ng
highest accuracy1-2µg
~100pmol of 20b oligo
0.2-0.5µgBR: 30-100ng
HS: 2-10ng
RNA: 10-50ng
BR: 100-500pg
HS: 10-50pg
EtBr: 50-200ngDNA: 0.5-50ng
RNA: 25-250ng
DNA: 0.5–50ng
RNA 6000 Nano: 25-250ng
RNA 6000 Pico: 0.5–5ng
price per assayonly hand work0.3-1.2€0.3-2¢low res.: ~3¢
high res.: ~30¢
>15¢>1.6€



  1. 1,0 1,1 high (~30%) measurement-to-measurement variability



NB! Normally, there is no restriction for using concentration-measurement devices. All of them should be handled as "dirty" (contaminated by other NA) devices.


  • accurate pipetting is critically important for accurate measurements:
» be sure, that sample is well mixed:
⇒ be sure, that sample is dissolved completely
⇒ NA may be freezed out from the solution (especially without snap freezing): mix solution after melting
⇒ use special precautions for pipetting of viscous solutions
» minimal (still accurate) sample volumes for different pipettes:
pipetteminimal volume
2µl0.8µl
10µl2µl
200µl10µl
1ml200µl
» check, that there is no drops/liquid on the outer surface of the tip
» do not use more than 50x dilutions. For preparation of seriously diluted samples use sequential dilutions: combination of 40x and 50x dilutions instead of one 2000x dilution.
» pipetting of small volumes may be controlled on high-precision balance


NB! Quantitation should NOT be very accurate for most of applications. To save sample ESTIMATE concentration instead of accuratly measure it. ~0.3µl of solution (2µl pipette) is accurate enough for estimation.


  • there is no "the best of the best" protocol. Different procedures have different advantages



[править] UV adsorbtion


advantagesdisadvantages
  • cheap
  • fast
  • accurate measurement of oligonucleotide concentration
  • does not require regular calibration
  • spectrum curve provides limited information about purity
  • low sensitivity
  • sensitive to contaminations (proteins, other NA, phenol)
  • unable to distinguish ssDNA // dsDNA // RNA


» if compare with normal spectrophotometer, Nanodrop is:
⇒ faster
⇒ more sensitive



[править] Intercalator fluorescence


advantagesdisadvantages
  • highest sensitivity
  • fast
  • can distinguish ssDNA // dsDNA // RNA
  • require regular calibration
  • unstable, water sensitive intercalator dye
  • sensitive to specific contaminations
  • sensitive to low-molecular weight NA's


[править] Comparison of Fluoro-NanoDrop and Qubit

  • Fluoro-NanoDrop:
» is ~10-100x cheaper if compare with Qubit
» measurements are less stable: ~30% for NanoDrop, ~15% for Qubit
» provides fluorescent spectrum. It is possible to distinguish unpurity of the sample
  • Fluoro-NanoDrop is more sensitive, than Qubit. Detection ranges of Fluoro-NanoDrop and Qubit overlap slightly for BR ans HS kits:
kitFluoro-NanoDropQubit
dsDNA BR10pg - 5ng3ng - 1µg
dsDNA BR1pg - 0.5ng0.2-100ng



[править] Size selection and interc. fluorescence

advantagesdisadvantages
  • with size-selection it is possible to be sure, that quantitation is not influenced by other factors
  • analysis of NA degradation
  • using special intercalator it is possible to distinguish ssDNA // dsDNA // RNA
  • long process
  • expensive if compare with other methods


» sensitivity is between "UV adsorbtion" and "Intercalator fluorescence"
» electrophoresis is a dirty procedure
» normally, capillary electrophoresis has a smaller size range, than normal electrophoresis
» capillary electrophoresis gives reproducible, digital data



Источник — «http://molbiol.ru/wiki/NA_quantitation»


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