Hi all,
I have been using UV spectrometery for DNA quantification. Where is it limited to use within a certain OD range, though I get reliable repeatability with that. It doesn't support the minute concentrations of DNA to quantify reliably.
Please suggest a good and cheap way to quantify DNA in a reliable manner.
Qubit Fluorometer may help you.... check this link...
http://www.lifetechnologies.com/in/en/home/life-science/laboratory-instruments/fluorometers/qubit/qubit-fluorometer/qubit-vs-competitors.html
Dear Lakshmi,
most accurate way - qPCR approach (Quantifiler® Human DNA Quantification Kit et al)
a good and cheapest way - Qubit 2.0 or 3.0
I agree with Vladimir that the qPCR is the best way to quantify the DNA when present in very low amount.
I personally don't like the Qubit, even if it is becomming a gold standard prior performing Next Gen Sequencing. the reproducibility is not that high considering that the reagent are hyper light sensitive and can be affected by the light in the room...
Another method to consider is the picogreen.
Nanodrop is nice when your DNA are in sufficient amount but lose in reliability for low concentration (below 75ng/ul).
Best of luck,
qPCR is very accurate, but qubit in my experience is actually very good. And generally good enough. And really quick compared to qPCR (depending on bulk sample number). As mentioned by Ludovic, nanodrop is completely useless at low concentrations.
Best,
Vegard
you can use nano drop to see the concentrations of DNA by using one micro liter of sample. But it will be costlier than spectrophotometer.
all the best
Nanodrop gives an indicative concentration and isn't efficient for low concentration.
By gel electrophhoresis using a known amount of DNA of the same size
The limit of the eye using BrEt staining is 3ng
Nanodrop 2200 seems to give fairly accurate and precise measurements. Quicker than qPCR, although I have not quantified the same sample using both approaches, so I cannot compare the two.
PicoGreen assays are much more sensitive than any OD260-based method and more accurate in that they are not as heavily influenced by free nucleotides, dinucleotides and other short degradation products. Invitrogen's kit Quant-IT works well.
Thank y'all for ur valuable answers.
Is there a scope of identifying the level of purity and carryover salt content in the DNA sample using picogreen assays?.
My requirement is to analyze plant genomic DNA, assessment of same for its quality and quantity.
Can it be possible with picogreen?
Please suggest
Qubit will give you a more reliable concentration of your DNA. Quant-it is its former name and I think it's been discontinued. You will need the equipment though and it will not give you the purity. On the other hand, nanodrop will give you purity, but it will also read other contaminants. If you don't have any specific equipments, just run your DNA on a gel and compare with known samples or with DNA mass ladder.
Qubit is a picogreen-based fluorimeter sold by Life technologies. Quant-iT kits are still available. Sandra is correct that picogreen assays do not provide purity estimates. A260/280/230 readings can do that. The use of any of the particular methods suggested here depend on what level of accuracy you require. Gel image estimates are not as accurate as UV spectorphotometry or Picogreen assays. On the other hand, provided you already have a spectrophotometer (as you indicate you do), gels and Picogreen assays are more expensive than UV spectrophotometry, so you will have to balance cost, ease, and accuracy in choosing a method. Picogreen assays are the most expensive of the methods discussed here but should be used for applications that require accurate quantification, such as nextgen sequencing library preps, etc.
Many thanks for your advise,
My requirement is not nexgen sequencing but for absolute quantification (standard curve) of a GMO event by qPCR.
For this purpose and to be precise in analysis we are looking for better option to UV spec to assess the quality and quantity of DNA solution after each preparation.
Dear Dr Lakshmi Narayana Lavu,
I would like to thank you for your question.
Highly accurate and cheap way to measure genomic DNA is with gel electrophoresis and nano drop machine.
NanoDrop instruments measure nucleic acid concentrations using only 1µL of sample.
All NanoDrop instruments utilize a patented sample retention system that allows the quantification of DNA and RNA from 1 - 2 µL samples.
NanoDrop 2000c UV-Vis Spectrophotometer
NanoDrop 3300 Fluorospectrometer
Technical resources: Assessment of Nucleic Acids Prior to qPCR and RT-qPCR
with best regards
Khaled
cDROP (drop sense 96) is the cheapest, fastest and very accurate. Best part is that it gives you A260/260/230 and a wealth of other info about the sample.
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