Recommended to use Qubit when you think accuracy matters, or when NanoDrop gives a reading of <50 ng/Ál. Electrophoresis should be used anytime trying to quantitate subspecies of DNA.

Overview

The lab currently has 3 ways of determining DNA concentration available: Qubit, NanoDrop, and Agarose gel electrophoresis. Each of these methods has advantages and disadvantages as follows:

Method Advantages Disadvantages
Qubit Ability to detect lowest concentrations Dilution required to determine concentrations.(Instrument calculates stock concentration)
Typical Range: (ng/Ál) Highly tolerant of salts, solvents, and RNA contamination Requires creation of 2 point standard curve so a bit annoying if only have a single sample
BR: 1-100 HS: 0.05-60 Most accurate method(accurate within 10% based on single dilution curve)í Takes slightly longer than NanoDrop
NanoDrop Fastest method Instrument not in our lab
Reported Range: Largest range of acceptable concentrations Not as accurate as Qubit(error rate of 40 - 160% below 30ng/Ál based on single dilution curve)í
5-3000 ng/Ál Requires only 1 Ál of sample Salt, RNA, and Solvents all alter the apparent concentration
Gel Salts, solvents, and RNA play no role in determining concentration Requires estimation of concentration based on similar banding pattern to ladder
Allows concentration determination of specific subspecies. Requires previous use of other method or outright guess to know how much sample to load
  Most time consuming
  low percentage agarose gels are necessary for identifying genomic DNA which is also highly subjective to diffusion
ísee attached excel graph near bottom of page for details

Qubit

  • Full manual can be found here.
  • Two main protocols exist for determining DNA concentration Broad Range (BR) and High Sensitivity.
    • The protocols are identical, but require the use of different buffer, dye, and standards.
    • The correct protocol to use will be based on expected concentration, but for most work, BR is appropriate.
  • Between 1 and 20 Ál of sample is required, with 2 Ál being the amount typically used by the lab.

BR Protocol

  • With this assay, the instrument can read diluted concentrations between 10pg/Ál and 10ng/Ál.
    • Maximum detection window: 0.1ng/Ál to 2,000ng/Ál (with decreased confidence above 1,000ng/Ál) for 20Ál and 1Ál sample respectively.
    • Typical detection window: 1ng/Ál to 1,000ng/Ál (with decreased confidence above 500ng/Ál) for 2Ál of sample.

  1. Make working stock by diluting 1Ál of BR dye into 199Ál dsDNA BR Buffer for each sample and standard.
    • be sure to make enough working stop for all your samples plus 2 standards.
  2. Aliquot 190 Ál working stock into Qubit tubes for both standards.
  3. Aliquot 198 Ál working stock into Qubit tubes for each sample.
  4. Add 10 Ál of appropriate standard to each standard tube. Briefly vortex after adding.
    ALERT! Should be done before samples to ensure at least 3 minute incubation at RT
  5. Add 2 Ál of each sample to each sample tube. Briefly votrex after adding.
  6. On the instrument select home on the bottom left, then select dsDNA BR assay, and yes to reading new standards
    TIP If intending to transfer data to memory card, it is best to clear the data first by selecting data on the bottom right and then clear data.
  7. Follow on screan instructions to read both standards and first sample.
    ALERT! Temperature affects the assay, so avoid warming the tubes by holding them excessively in your hands prior to placing them in the instrument, and select 'read' quickly after
    1. After first sample is read, the units and dilution factor can be entered by selecting 'calculate stock concentration'
    2. selecting 'save' will allow easy transfer to a usb memory card after all samples are read.
  8. Continue reading all samples.
  9. If transferring to memory card, select Data from bottom right, memory stick icon will have a green circle next to it, select all data to be transferred (check box top left can be very helpful) and click the usb memory card icon.

HS Protocol

  • With this assay, the instrument can read diluted concentrations between 1pg/Ál and 500pg/Ál.
    • Maximum detection window: 0.005ng/Ál to 120ng/Ál (with decreased confidence below 0.01ng/Ál and above 100ng/Ál) for 20Ál and 1Ál sample respectively.
    • Typical detection window: 0.05ng/Ál to 60ng/Ál (with decreased confidence below 0.1ng/Ál above 50ng/Ál) for 2Ál of sample.
  1. Same protocol as BR, just substitute HS dye, and dsDNA HS buffer for the working stock, and be sure to use the HS standards instead of the BR standards

NanoDrop

  • 1 Ál of sample required, capable of determing concentration between 5ng/Ál and 3,000ng/Ál according to manufacturer
    • 1Ál vs 2Ál of sample does not seem to make a difference in readings (see below)
  • Concentration determination is affected by salts, solvents, and RNA present in the sample.
    • Looking at 260/280 and 260/230 ratios can give idea of contamination from these sources, but ratios only indicate problem.

NanoDrop Protocol

  1. Take samples, pippette tips, and tube of blank solution (typically water or elution buffer depending on what your DNA was eluted in) across hall in MBB to DNA sequencing core, and in the Zhang lab in WEL
  2. Log into computer and rinse pedestal and arm with di Water. Dry with kimwipes.
    • In MBB login information is your personal eid and password.
    • WEL requires no logon information, but does require a key to the Zhang lab.
  3. Initialize the NanoDrop 1000 software by double clicking on the NanoDrop icon.
  4. Select nucleic acid
  5. Pipette 1Ál liquid from blank tube onto pedestal and lower the arm.
  6. Hit 'ok' on computer to blank the instrument
  7. Use kimwipe to dry the pedestal. Reload a fresh 1Ál aliquot from blank tube onto pedestal, and lower arm back down.
  8. Select Re-Blank from the top left of the window.
  9. Use kimwipe to dry pedestal. Load 1Ál of sample onto pedestal and lower the arm.
  10. Enter sample name on the right hand side of the window, and select 'measure' on the top left.
  11. Inspect the curve to ensure it consists of a high reading on the left, a dip, another peak, and then a slope to the baseline.
  12. Record concentration of sample.
    TIP if determining multiple samples, you can wait till the end and use the 'report - show report - save report - save as csv table' option to have a record of all concentrations as well as 260/280 260/230 ratios.
  13. Repeat drying and loading of samples till done.

Agarose Gel Electrophoresis

  • Capable of estimating DNA concentration between 125 and 30 ng irrespective of salts and solvents.
  • Detection based on actual ng, not on ng/Ál.
  • Only method capable of determining concentration of 1 sized subspecies in mixture of different sizes.

Agarose Gel Electrophoresis Protocol

  1. Pour agarose gel.
    • Percentage of the gel should be based on range of linear separation. (see table below)
  2. Be sure to use marker with known amounts of each size marker (for instance the 1kb NEB marker)
  3. Load different amounts of DNA such that you estimate that at least 1 well has between 30ng and 125ng of DNA.
  4. Run gel till achieve good separation of marker.
  5. Take image of gel on imager.
    ALERT! Remember that low percentage gels will diffuse quickly necessitating quick return to electrophoresis.
  6. Estimate amount of DNA in each well as compared to known quantities of different bands.
  7. Determine concentration of DNA based on number of Ál loaded in each well

Gel Percent Range of separation (kb)
0.3 5-60
0.6 1-20
0.7 0.8-10
0.9 0.5-7
1.2 0.4-6
1.5 0.2-3
2.0 0.1-2

Comparisons

This section contains information regarding the comparison of the different protocols to assess their limitations.

  • Invitrogen Genomic DNA prep of REL606:
    1. 4 1ml aliquots of an overnight culture were isolated and eluted in 2 25Ál elution buffer and pooled together to hopefully eliminate some variation of preparation.
    2. Qubit and NanoDrop readings were taken in triplicate for 2Ál and 1Ál aliquots with the following results:
      1. Qubit 2Ál is the most accurate reading.
      2. Both methods capable of determining concentration at this level with little difference.
      3. Readings:
Method Measurements (ng/Ál) Average ng/Ál Standard Deviation
Qubit 1Ál 121, 125, 127 124.3 3.05
Qubit 2Ál 125, 125, 124 124.3 0.57
NanoDrop 1Ál 125.44, 128.5, 128.04 127.3 1.65
NanoDrop 2Ál 128.82, 127.39 (-)í 128.1 1.01
ímeasurement read -3.35ng/Ál, but this graph showed this reading to be bad, and was removed from analysis. It is included in this section to highlight the potential for error in reading a sample with the NanoDrop.

  • Accuracy of measurement across range of concentrations.
    1. 5 50% dilutions were made from the same Genomic DNA prep used in the previous experiment.
    2. Triplicate readings using 2Ál for Qubit and 1Ál for NanoDrop were used based on previous findings with the following results:
      1. Qubit was significantly (p < 0.05 by ttest between groups of measurements) more accurate (closer to the expected value) for 4 of 5 measurements.
      2. NanoDrop was non-signficantly (p = 0.058 by ttest) more accurate for 1 of 5 measurements.
      3. NanoDrop was unable to detect an expected DNA concentration of 3.9 ng/Ál (average reading of -2.34 ng/Ál). Should be noted that this is slightly outside the reported range of the instrument.
Serial_Dilution.jpg

  • Agarose Gel Electrophoresis estimation of concentration.
    1. Based on previous findings of ~125ng/Ál concentration, 1 Ál of original and 5 serial dilutions loaded onto 0.6% gel agarose gel.
    2. Run for approximately 1 hour with the following results:
      1. 125 ng well approximately matches the 125ng 3kb band in the NEB 1kb marker and is therefore in agreement with other methods. This assay would be much more difficult if actually used to estimate the concentration rather than confirm the concentration.
      2. "Good_Electrophoresis.pdf" represents image taken immediately after 60 minute run.
      3. "Bad_Electrophoresis.pdf" represents image taken after gel sat for 10minutes, run for an additional 15, and new picture taken.

  • Experiment still planned:
    • Spike excess salt and RNA into sample to see specific differences in NanoDrop and Qubit

-- Main.DanielDeatherage - 20 Aug 2012

RNA Quantification

This section reviews RNA quantification using both the Nanodrop and Qubit

  • RNA quantification is almost exactly identical to DNA quantification
  • Both instruments have specific RNA settings
    1. Nanodrop: "RNA-40"
    2. Qubit: "RNA"
      • "RNA Broad" is for RNA Broad Range ie. high-abundance (20-1000ng) RNA samples... we don't have this kit, but we could buy it

Method Advantages* Accurate Concentration Range Concentration Range if necessary
Nanodrop can measure 1ul 5-3,000 ng/ul 2-3,000 ng/ul
Qubit accuracy, very RNA-specific 250 pg/ul-100ug/ul 250pg/ul-100 ug/ul

*"Concentration Range if Necessary" isn't suggested, isn't very accurate, and gives a bad curve, BUT if you have very low concentration and very little sample you can use this to just verify RNA in your sample

  • If you have an idea of the RNA concentration (after Nanodrop), use the graph below ("RNA_Qubit.jpg") to determine how dilute your sample should be for the Qubit. Usually 2ul is best for almost all concentrations, but the less concentrated, the more sample you'll need to use.

-- Main.LindseyWolf - 4 Sept 2012

Topic attachments
I Attachment Action Size Date Who Comment
pdfpdf Bad_Electrophoresis.pdf manage 1731.2 K 24 Aug 2012 - 16:02 Main.DanielDeatherage Gad Electrophoresis image
jpgjpg Bad_NanoDrop_output.jpg manage 68.0 K 23 Aug 2012 - 19:12 Main.DanielDeatherage Bad NanoDrop Graph
pdfpdf Good_Electrophoresis.pdf manage 1650.7 K 24 Aug 2012 - 16:01 Main.DanielDeatherage Good Electrophoresis image
jpgjpg Good_NanoDrop_output.jpg manage 72.5 K 23 Aug 2012 - 19:12 Main.DanielDeatherage good Nanodrop Graph
jpgjpg RNA_qubit.jpg manage 27.2 K 04 Sep 2012 - 17:18 Main.LindseyWolf  
jpgjpg Serial_Dilution.jpg manage 26.5 K 23 Aug 2012 - 17:00 Main.DanielDeatherage results graph of serial dilution experiment comparing Qubit and Nanodrop
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Topic revision: r12 - 04 Sep 2012 - 17:56:35 - Main.LindseyWolf
 
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