After extracting DNA, RNA or protein from a biological entity, you often want to measure just
how much you have. Because you need to decide how much to use in your other essays; whether you're
going to use it for proteomics or for Western blot or if it's DNA and RNA maybe you want to
use it for polymerase Chain Reaction (PCR) or to sequence it and find out what bases form that
particular nucleic acid. And so quantification of your biological sample that you've extracted
from a cell or from tissue or from any biological entity is part of the workflow in the laboratory.
there are two common Optical based technologies that can be used to quantify nucleic acids and
nucleic acids we're always referring to DNA or RNA. for example using a spectrophotometer you
can select a specific wavelength where most proteins will have a residue that absorbs at
that wavelength so if you... for proteins you select a 280 nanometers for instance you will
have the capability to measure how much protein you have, because most proteins contain the amino
acid residue tryptophan which absorbs at 280 nanometers so it can be a surrogate for how much
protein you have. For nucleic acids absorbance at 260 nanometers if you're using a spectrophotometer
with no dyes added to your sample, is a great place to start because most nucleic acids will
absorb and they absorb within a range but 260 nanometers is a good indicator of how much nucleic
acids are in the sample absorbance just based on wavelength specific wavelength option is usually
not that accurate because you can have interfering compounds that also absorb at that wavelength and
so it can inflate how much you actually have. Or you may have contaminants that interfere with how
much is actually in there and so the alternative is often to use a fluorescence-based measurement.
In fluorescence-based measurements you use a dye (flurohore) that attaches to the biological
molecule. So if we're talking about DNA a specific dye (flurophore) for DNA will be added to the
sample or if we're talking out about RNA then a specific dye for RNA will be added to the sample;
or if protein likewise a specific dye for protein would be added to the sample. And in so doing,
you're moving away from in... from indiscriminate measuring to specific measuring because now you're
going to... the dye will attach itself to the particular biological molecule that you're
measuring and then you're going to measure the fluorescent signal from that dye as an
indirect way of specifically measuring how much of the biological molecule you have.
The qubit is one such so-called fluorometer in the sense that you're using the fluorescence molecule
and so it measures fluorescence read out for you. And to use it, here are the steps you follow; if
you suspect that you have a lot of the biological molecule in your sample, so for instance if you
extracted DNA from a biological Source where there is a lot of DNA, then you you may want
to use a specialized kit such as the broad range kit which means you know you're expecting quite a
high amount of the DNA or RNA or whatever protein. But if you're extracting from a sample Source or a
biological Source where you know that there isn't much of it, then you can use a kit such as a high
sensitivity kit and in this you can be you can have more sensitivity for lower concentrations
of the biological molecule you're measuring. So using the qubit (a fluorometer) you can get qubit
double-stranded DNA High sensitivity assays or you can get the double stranded DNA broad range
assay. You may also be measuring a single... a single stranded DNA system, so then you can get
the single stranded DNA assay. If you have RNA, you can get specific RNA assay and if
you have a lot of RNA you can get the RNA broad range assay and they also have a protein assay.
Step one is to dilute the fluorescent dye that the supplier gives to you. The fluorescent dye
is 200 times concentrated and you want to dilute it, so that it's at 1x for the actual measurement.
And so to do this you put one part of the dye to 199 parts of the diluent that they provide. So
let's say for example if you're going to make 400 microliters total for the number of samples that
you have, then you're going to put 200 microliters of the dye into 398 microliters of the diluent
that forms the working stock or Master mix that you can now use to dilute your sample into. And so
when your sample comes into contact with this dye it's going to... the dye is going to intercalate
with the biological molecule and it's going to fluoresce very strongly when you put it into the
qubit instrument. And that way you'll be able to measure how much of the biological molecule
you have. So if you're using something like the high sensitivity kit where you don't expect to
have a lot of analyte, then you can do a sample dilution into the dye within the range of say
one in 20 to maybe one in 100 (if you feel like you you may have quite a bit of analyte there).
So basically you want to dilute the sample with the dye that you've made up because you
need the reagent to interact with your sample so you can measure it
the next step is then to just Vortex the sample to mix it and then just let it sit for about two
minutes to allow the dye to intercalate with the biological molecule now you are ready to read the
acid to get the readout very simple but just when you go to the instrument you're going to need to
select the assay type that you are using whether you're doing a DNA double-stranded essay or you're
doing a single stranded DNA or maybe you're doing RNA or protein just be specific and select that
then you're going to place the tube that this apply you with actually the tube that contains
your sample and you're going to close the lid of the instrument then you're going to hit read to
get your readout and because the instrument is already programmed to factor in that you would
have done a dilution it will ask you how much of your sample you place into the die and so
the reader is actually your final concentration and that's it that's how easily you can use the
qubit to fluorometrically... fluorometrically measure how much biological molecules you have
in your sample. So yes thank you for checking out this video and all the best with your measurement.