(r13) Barrick Lab :: DNAConcentrationDetermination

---+!!! Methods for Determining the Concentration of DNA Samples
This page explains the most commonly used methods for measuring DNA (or RNA) concentrations and the advantages and disadvantages of each one.

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---+Overview

|*Method*|*Advantages*|*Disadvantages*|
|*Qubit*|Ability to detect lowest concentrations|Dilution required to determine concentrations.(Instrument calculates stock concentration)|
|Typical Range: (ng/µl) |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)¡|Takes slightly longer than !NanoDrop|
|*Method*|*Advantages*|*Disadvantages*|
|* !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/µl based on single dilution curve)¡|
|5-3000 ng/µl |Requires only 1 µl of sample|Salt, RNA, and Solvents all alter the apparent concentration|
|*Method*|*Advantages*|*Disadvantages*|
|*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|

---++ %ICON{choice-yes}% Recommended Method: Qubit 2.0 Fluorometer

The Qubit spectrophotometer uses a DNA dye whose fluorescence changes upon DNA (or DNA binding). It relies on reading two standards every time that you make measurements for calibration. Two main protocols exist for determining DNA concentration, depending on whether you want to be able to measure samples over a *Broad Range (BR)* or with a high *High Sensitivity* (for lower DNA concentrations).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. We have a Qubit 2.0 unit in both the MBB and WEL labs.

*Sample Assumptions:*
   * You have double-stranded DNA. %BR% Other Qubit kits must be used to accurately determine the concentrations of single-stranded DNA or RNA because they interact with the fluorescent dyes differently.
   * From 1 to 20 µl of DNA sample is required, with 2 µl being the amount typically used by the lab.

*Additional Resources*
   * [[https://www.thermofisher.com/us/en/home/life-science/laboratory-instruments/fluorometers/qubit/qubit-assays.html][Main Qubit Instrument and Assay Page]]
   * [[https://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/cell-tissue-analysis/qubit-all-file-types.par.0519.file.dat/qubit-2-fluorometer-user-manual.pdf][Link to the Qubit manual]]
   * [[https://www.youtube.com/watch?v=RRKZN--7jqg][Video discussing the advantages of the Qubit and how to perform an assay (from Thermo Fisher)]]

---+++ *BR* (Broad Range) Protocol
   * With this assay, the instrument can read *diluted concentrations* between 10pg/µl and 10ng/µl.
      * Maximum detection window: 0.1ng/µl to 2,000ng/µl (with decreased confidence above 1,000ng/µl) for 20µl and 1µl sample respectively.
      * Typical detection window: 1ng/µl to 1,000ng/µl (with decreased confidence above 500ng/µl) for 2µl of sample.

   1. Make working stock by diluting 1µl of BR dye into 199µl dsDNA BR Buffer for each sample and standard.
      * be sure to make enough working stop for all your samples plus 2 standards.
   1. Aliquot 190 µl working stock into Qubit tubes for both standards.
   1. Aliquot 198 µl working stock into Qubit tubes for each sample.
   1. Add 10 µl of appropriate standard to each standard tube. Briefly vortex after adding.
      %BR% %X% _Should be done before samples to ensure at least 3 minute incubation at RT_
   1. Add 2 µl of each sample to each sample tube. Briefly votrex after adding.
   1. On the instrument select home on the bottom left, then select dsDNA BR assay, and yes to reading new standards
      %BR% %T% _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._
   1. Follow on-screen instructions to read both standards and first sample.
      %BR% %X% 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'
      1. selecting 'save' will allow easy transfer to a usb memory card after all samples are read.
   1. Continue reading all samples.
   1. 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* (High Sensitivity) Protocol
   * With this assay, the instrument can read *diluted concentrations* in the range 1-500pg/µl.
      * Maximum detection window: 0.005ng/µl to 120ng/µl (with decreased confidence below 0.01ng/µl *and* above 100ng/µl) for 20µl and 1µl sample respectively.
      * Typical detection window: 0.05ng/µl to 60ng/µl (with decreased confidence below 0.1ng/µl above 50ng/µl) for 2µl 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 µl of sample required, capable of determing concentration between 5ng/µl and 3,000ng/µl according to manufacturer
      * 1µl vs 2µl 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
   1. 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._
   1. Initialize the !NanoDrop 1000 software by double clicking on the !NanoDrop icon.
   1. Select nucleic acid
   1. Pipette 1µl liquid from blank tube onto pedestal and lower the arm. 
   1. Hit 'ok' on computer to blank the instrument
   1. Use kimwipe to dry the pedestal. Reload a fresh 1µl aliquot from blank tube onto pedestal, and lower arm back down.
   1. Select Re-Blank from the top left of the window.
   1. Use kimwipe to dry pedestal. Load 1µl of sample onto pedestal and lower the arm.
   1. Enter sample name on the right hand side of the window, and select 'measure' on the top left.
   1. 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.
   1. Record concentration of sample.
      %BR% %T% 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.
   1. 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/µl.
   * 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)
   1. Be sure to use marker with known amounts of each size marker (for instance the 1kb NEB marker)
   1. Load different amounts of DNA such that you estimate that at least 1 well has between 30ng and 125ng of DNA.
   1. Run gel till achieve good separation of marker.
   1. Take image of gel on imager.
      %BR% %X% Remember that low percentage gels will diffuse quickly necessitating quick return to electrophoresis.
   1. Estimate amount of DNA in each well as compared to known quantities of different bands.
   1. Determine concentration of DNA based on number of µl 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 25µl elution buffer and pooled together to hopefully eliminate some variation of preparation.
      1. Qubit and !NanoDrop readings were taken in triplicate for 2µl and 1µl aliquots with the following results:
         1. Qubit 2µl is the most accurate reading.
         1. Both methods capable of determining concentration at this level with little difference.
         1. Readings:
|*Method*|*Measurements (ng/µl)*|*Average ng/µl*|*Standard Deviation*|
|Qubit 1µl | 121, 125, 127| 124.3| 3.05|
|Qubit 2µl | 125, 125, 124| 124.3| 0.57|
|!NanoDrop 1µl| 125.44, 128.5, 128.04|127.3|1.65|
|!NanoDrop 2µl| 128.82, 127.39 (-)¡|128.1|1.01|
¡measurement read -3.35ng/µl, 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.
      1. Triplicate readings using 2µl for Qubit and 1µl 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.
         1. !NanoDrop was non-signficantly (p = 0.058 by ttest) more accurate for 1 of 5 measurements. 
         1. !NanoDrop was unable to detect an expected DNA concentration of 3.9 ng/µl (average reading of -2.34 ng/µl). Should be noted that this is slightly outside the reported range of the instrument.
%ATTACHURL%/Serial_Dilution.jpg

   * Agarose Gel Electrophoresis estimation of concentration.
      1. Based on previous findings of ~125ng/µl concentration, 1 µl of original and 5 serial dilutions loaded onto 0.6% gel agarose gel.
      1. 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.
         1. "Good_Electrophoresis.pdf" represents image taken immediately after 60 minute run.
         1. "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"
      1. 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
Attachments
Topic attachments
I	Attachment	History	Action	Size	Date	Who	Comment
pdf	Bad_Electrophoresis.pdf	r1	manage	1731.2 K	2012-08-24 - 16:02	DanielDeatherage	Gad Electrophoresis image
jpg	Bad_NanoDrop_output.jpg	r1	manage	68.0 K	2012-08-23 - 19:12	DanielDeatherage	Bad NanoDrop Graph
pdf	Good_Electrophoresis.pdf	r1	manage	1650.7 K	2012-08-24 - 16:01	DanielDeatherage	Good Electrophoresis image
jpg	Good_NanoDrop_output.jpg	r1	manage	72.5 K	2012-08-23 - 19:12	DanielDeatherage	good Nanodrop Graph
jpg	RNA_qubit.jpg	r1	manage	27.2 K	2012-09-04 - 17:18	LindseyWolf
jpg	Serial_Dilution.jpg	r1	manage	26.5 K	2012-08-23 - 17:00	DanielDeatherage	results graph of serial dilution experiment comparing Qubit and Nanodrop
Barrick Lab > ProtocolList > DNAConcentrationDetermination
Contributors to this topic
DanielDeatherage, JeffreyBarrick, KateElston, JuliePerreau, VictorLi, IsaacGifford, LindseyWolf, PengGeng
Topic revision: r13 - 2016-02-16 - 17:51:17 - Main.JeffreyBarrick