Intercalator fluorescence

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problems/questions

 Problem list:

  • Qubit & computer
  • Qubit and NanoDrop control samples
  • typical standard fluorescences for control samples


Mol.biology // Next-generation sequencing // NA quantitation

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Содержание


[править] Protocol for Qubit

  1. number of measurements/tubes:
       » number of samples:
       » 2 standards
       » 1 control
  2. prepare WS (Working Solution):
  3. sign lids of Qubit assay tubes;
  4. (optional) set up standards:
    » st.1: 190µl WS + 10µl of Comp.C
    » st.2: 190µl WS + 10µl of Comp.D
  5. (optional) set up control: 10µl control + 190µl WS;
  6. set up samples: (1-20)µl sample + up to 200µl WS;
  7. vortex and spin down the tubes;
  8. be sure, that all assay tubes have normal temperature;
  9. leave for 2-5 minutes at RT[1];
  10. perform the concentration measurement on Qubit™ fluorometer:
    • press any key to turn on the fluorometer
    • press HOME
    • highlight the name of the kit ⇒ GO
    • select:
    » "Run new calibration" ⇒ GO
    » "Use last calibration" ⇒ GO
    • perform measurements following instructions on the screen:
    ▫ insert Standard 1 and press GO
    ▫ insert Standard 2 and press GO
    ▫ insert Sample 1 and press GO ⇒ record values;
  11. calculate concentration[2]



[править] Protocol for NanoDrop 3300 Fluorospectrometer

  1. number of measurements/tubes:
       » number of samples:
       » 2 standards
       » 1 control
  2. prepare 2xWS (2x Working Solution):
  3. prepare standards (mix by pipetting):
    » st.1: 1µl 2xWS + 1µl of Comp.C
    » st.2: 1µl 2xWS + 1µl of Comp.D
  4. set up samples: 1µl sample + 1µl 2xWS;
  5. leave for 2-5 minutes at RT[1];
  6. perform fluorescence measurement on Nanodrop 3300 fluorometer;
  7. calculate concentration[3]



[править] Notes

 QubitNanoDrop 3300 Fluorospectrometer
detection rangeBR: 3ng - 1µg
HS: 0.2-100ng
RNA: 5-100ng
BR: 10pg - 5ng
HS: 1-500pg
highest accuracyBR: 30-100ng
HS: 2-10ng
RNA: 10-50ng
BR: 100-500pg
HS: 10-50pg
price per assay0.3-1.2€0.3-2¢


  • special clear tubes are used for Qubit measurements, we use Axygen PCR-05-C (VWR #10011-830)[4]


  • HS and BR assays: aliquot kit components upon arrival and store them at -20°C; working aliquots are kept at +4°C


  • components of Quant-iT assays:
ComponentKit
dsDNA HSdsDNA BR
A200x dye concentrate in DMSO
Bdye dilution buffer
CTE buffer
DdsDNA, 10ng/µl in TEdsDNA, 100ng/µl in TE


[править] Intercalator dye

intercalator dye is unstable and water sensitive:

  • should be aliquoted in tightly-closed tubes
  • do not open untill tube reaches room temperature
  • tips for dispensing should be dry



[править] Control NA solution

  • fluorescence measurement series may vary by up to ~30%. It is necessary to prepare control NA solution and measure it along with other samples to estimate the deviation of a particular series. Prepare in large quantity and make aliquots. For dsDNA measurements the stock solution is a plasmid DNA (w/o RNA), 50ng/µl.
» Qubit, BR: 50ng/µl, pDNA stock
» Qubit, HS: 5ng/µl, fresly 10x diluted (4.5µl TE + 0.5µl pDNA stock)
» Fluoro-NanoDrop, BR: 250pg/µl, freshly 200x diluted (99.5µl TE + 0.5µl pDNA stock)
» Fluoro-NanoDrop, HS: 25pg/µl, freshly 2000x diluted
▫ I dilution, 40x: 19.5µl TE + 0.5µl pDNA stock
▫ II dilution, 50x: 24.5µl TE + 0.5µl I dilution



[править] Assay accuracy

  • photobleaching is negligible for Qubit (no data for Fluoro-NanoDrop):
» <0.3% after 9 readings
» <2.5% after 40 readings


  • strongly fluorescent materials may influence the assay. In laboratory:
» cellulose dust from paper, paper towels
» markers
» detergents, cleaning solutions


  • signal stability:
» HS and BR assays: ~3h. For estimation of concentration even next-day measurements are acceptable.
» ssDNA assay: 30min.


  • fluorescence drops with temperature:
» ~2% per 1°C for dsDNA HS assay
» ~5.3% per 1°C for dsDNA BR assay

To minimize temperature fluctuations:

» ensure, that all reagents have room temperature during assay
» measure fluorescence immediately after insertion tube into Qubit


  • effect of contaminants on Quant-iT assays:
ContaminantFinal concentration in the assay
dsDNA HSdsDNA BR
NaCl50mM10mM[5]
MgCl25mM[5]2mM[5]
AcONa30mM10mM
NH4Ac50mM10mM[5]
K phosphate, pH7.4-5mM[5]
Ethanol1%1%
Phenol0.1%[5]0.1%
Chloroform1%0.2%
SDS0.01%0.01%
Triton X-1000.01%[5]0.001%[5]
dNTPs0.1mM0.1mM
BSA10µg/µl[5]20µg/µl[5]
IgG0.5µg/µl10µg/ml
RNA1x6x
ssDNA1x1x
oligos1x3x



[править] Comparison of Fluoro-NanoDrop and Qubit

Factors, important for selection of machine:

» price: Fluoro-NanoDrop is ~10-100x cheaper if compare with Qubit
» accuracy: Fluoro-NanoDrop measurements are less stable: ~30% for NanoDrop, ~15% for Qubit
» sensitivity: Fluoro-NanoDrop is more sensitive, than Qubit. Detection ranges of Fluoro-NanoDrop and Qubit overlap slightly for both BR ans HS kits. Too concentrated samples SHOULD BE DILUTED before Fluoro-NanoDrop analysis.
kitFluoro-NanoDropQubit
dsDNA BR10pg - 5ng3ng - 1µg
dsDNA BR1pg - 0.5ng0.2-100ng


  • Fluoro-NanoDrop provides fluorescent spectrum. It is possible to distinguish unpurity of the sample




  1. 1,0 1,1 measurements right after preparing the sample and in an hour might be up to 30% different
  2. The value given by the Qubit corresponds to the concentration of sample in the assay tube (200µl). If sample volume is 1, 2, 3, 4, 5, 10, 15 or 20µl it is possible to use "Calculate sample concentration" option on Qubit. Otherwise, concentration should be recalculated manually: concsample = concQubit value x (200[µl]/Vsample[µl])
  3. take "st.1" fluorescence as a background: concsample = (Fsample - Fst.1) / (Fst.2 - Fst.1) x concst.2
  4. Invitrogen, #Q32856
  5. 5,0 5,1 5,2 5,3 5,4 5,5 5,6 5,7 5,8 5,9 acceptable result, but it would be better to add the same amount of contaminant to standard samples


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