General use instructions
Qubit DNA quantification machine from Invitrogen/Lifetech.
This is a fluorimetric means of quantifying absolute DNA amounts. For projects which critically rely on DNA quality, such as RADs and whole genome resequencing, this is the way to go for DNA quantifications.
The Qubit is on the bench of lab 204.
Please talk to your supervisor about the (shared or not) use of its reagents.
You shall need:
500 µL thin-walled PCR tubes AND Qubit assay kits
Here is how to use it:
Make sure reagents are at room temperature. For now we put everything at 4°C, just because we do not anticipate too much use in the near future. Always keep the size standards at 4°C - these are DNA standards which decay much faster at warmer temperatures - the other buffers/reagents could also be stored at RT.
1) prepare tubes. Use the special thin-walled 500ul PCR tubes provided (drawer). You need a single tube for each sample plus two extra tubes (size standards)
2) prepare working solution: Mix 199ul of Qubit buffer with 1 ul of qubit reagent (this is the small tube with the orange liquid)
3) prepare the DNA standards: Mix 190ul of working solution with 10ul of standard #1. Do the same with standard #2. keep track of the standards - don't mix them up
4) prepare user samples. For this, mix 199ul of working solution and 1ul of your DNA prep.
5) mix every tube shortly by vortexing (2-3 seconds)
6) Incubate all tubes for 2 minutes at room temperature
7) then get ready to measure:
_Touch screen of the machine to turn it back on (or plug it back in)
_select DNA to measure
_then select the broad range (BR) dsDNA measurements (this is the current kit we have)
_put the tube with standard #1 in the machine and press measure
_put the tube with standard #2 in the machine and press measure
_now measure your samples one after another
_take notes of your DNA concentration or
_plug in the USB-key provided with the machine. THIS DEVICE IS FOR THE QUBIT ONLY. Save your results on the stick, transfer the data on your machine, and then clean the data from the stick. The USB stick also contains manuals for the machine etc. Have a look. RETURN THE USB KEY TO THE DRAWER WITH THE MACHINE SUPPLIES
*Time to measure*
Sample preparation and measurements are real fast. Including the 2min incubation step, 11 samples got measured with a standard in approx. 10min.
*How does it compare to Nanodrop results?*
The Qubit measures true ds-DNA quantities, while the Nanodrop also picks up signal from contaminants etc. Thus, Nanodrop-DNA quantifications are less precise, and exaggerated compared to the "true" DNA concentrations. Again, for most applications the Nanodrop is just fine.
Here are true values:
Nanodrop:
Sample ID User ID Date Time ng/ul A260 A280 260/280 260/230 Constant Cursor Pos. Cursor abs. 340 raw
A Default 23.11.2011 10:12 85.31 1.706 0.950 1.80 0.59 50.00 230 2.882 0.382
B Default 23.11.2011 10:12 80.78 1.616 0.891 1.81 0.61 50.00 230 2.634 0.252
C Default 23.11.2011 10:13 85.52 1.710 0.931 1.84 0.65 50.00 230 2.618 0.304
D Default 23.11.2011 10:13 84.94 1.699 0.943 1.80 0.76 50.00 230 2.231 0.156
E Default 23.11.2011 10:14 78.23 1.565 0.876 1.79 0.50 50.00 230 3.148 0.297
F Default 23.11.2011 10:14 73.83 1.477 0.806 1.83 0.57 50.00 230 2.585 0.226
G Default 23.11.2011 10:14 85.44 1.709 0.928 1.84 0.99 50.00 230 1.730 0.123
H Default 23.11.2011 10:15 83.02 1.660 0.893 1.86 0.81 50.00 230 2.050 0.152
I Default 23.11.2011 10:15 84.66 1.693 0.917 1.85 0.88 50.00 230 1.924 0.144
Qubit:
Name Date/Time Assay Conc. Units Stock Conc. Units Assay Type Sample Vol (µL) Dilution Factor Std 1 RFU Std 2 RFU Std 3 RFU Excitation Green RFU Far Red RFU
A 12:57:46 PM 2011-11-23 12:57:46 PM 0.459 µg/ml dsDNA BR 157.17 16101.92 BLUE 2012.32
B 12:58:22 PM 2011-11-23 12:58:22 PM 0.402 µg/ml dsDNA BR 157.17 16101.92 BLUE 1789.43
C 12:58:34 PM 2011-11-23 12:58:34 PM 0.434 µg/ml dsDNA BR 157.17 16101.92 BLUE 1913.37
D 12:58:51 PM 2011-11-23 12:58:51 PM 0.579 µg/ml dsDNA BR 157.17 16101.92 BLUE 2482.72
E 12:59:09 PM 2011-11-23 12:59:09 PM 0.341 µg/ml dsDNA BR 157.17 16101.92 BLUE 1547.39
F 12:59:22 PM 2011-11-23 12:59:22 PM 0.426 µg/ml dsDNA BR 157.17 16101.92 BLUE 1882.05
G 12:59:33 PM 2011-11-23 12:59:33 PM 0.411 µg/ml dsDNA BR 157.17 16101.92 BLUE 1824.95
H 12:59:43 PM 2011-11-23 12:59:43 PM 0.562 µg/ml dsDNA BR 157.17 16101.92 BLUE 2414.13
I 12:59:54 PM 2011-11-23 12:59:54 PM 0.584 µg/ml dsDNA BR 157.17 16101.92 BLUE 2499.46
# Given I used 2ul of DNA in 198ul of working solution for the measurement, a measurement of 0.584ug/ml on the Quibit corresponds to a stock DNA concentration of 58.4ng/ul. The qubit can calculate the concentration of the stock solution (i.e. your actual DNA preps.) for you.
# If you remeasure the same sample on the Qubit, you will find differences in the measurements of around 0.5ng/ul or less.
# Thus, true DNA concentrations appear to be roughly 1/2 to 2/3 of what the Nanodrop says. This is based on the analysis of these 9 samples only - and will always critically depends on the quality of your DNA preps.