I work in a biobank and we all use Nanodrop it is very useful to know the purity of your DNA but spectrophotomers gives you nucleotides mesure and even if your DNA is degraded you can obtain very nice result with Nanodrop so the best is Nanodrop quantification plus DNA integrity visualization on an agarose gel. Some people think the is fluorimetry with a commercial kit, for ex , Picogreen that gives you the measure of double strain DNA. For me nanodrop plus agorose gel visualization is the best choice, simple and cheap.
what level are you looking to accurately quantify the DNA to? nanogram level or lower? and how many microlitres of it (the stock) is available to you?
I think nanogram, not lower. and not more than 10 microlitres. to check DNA after PCR
Also, Qubit fluorometer kit can be used....
Here, the related links:
1. http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/Qubit.html
2. http://probes.invitrogen.com/media/pis/mp32857.pdf
3.http://www.interactivebioscience.cz/admin/_docs/Fluorescencni%20stanoveni%20koncentrace%20nukleovych%20kyselin_Heidi%20Onderkova.pdf
Gel quantifiaction is most accurate in combination with Nanodrop spectrophotometer. Gel gives you rough estimation and proof that you have DNA. Nanodrop is only for a fine tuning.
Hi, I thing both methods are good, nanodrop is easier to use, but gel electrophoresis is more accurate.
you can use nanodrop, it is very efficient to measure the DNA concentration!
Qubit system mentioned above is more accurate for DNA, especially for low concentrations. For some DNA preps, NanoDrop measurements can be 10X higher than they really are. Qubit can also use 1-2ul. Instead of using their fluorometer, we use our real-time PCR machine to measure fluorescence. It's a little more work than the NanoDrop, but if you need very accurate measurements, it's the way to go.
hey alexandra the nanodrop wins hands down based on popularity here :P however what are the instruments you have at your disposal?..also i prefer the nanodrop to gel electrophoresis based quantification because the nanodrop gives you readings at 260, 230 and 280 (some even have a scan range of 200-320)...so you also get valuable information regarding protein contamination and if you are using commercial extraction kits then salt contaminations like guanidium salts (chaotropic salts are present in high concentrations in the lysis buffers). Also if you are procuring a nanodrop, please choose the make carefully as some require only 1-2 ul for accurate measurements whereas others require as much as 7-9 ul per reading. and you will have to take a minimum of 2-3 reading regardless of which manufacturer's nanodrop you are using...the gel electrophoresis method is good if you are comparing 100s of ng of DNA but for lower amounts you will need the nanodrop..
hey
even i think nanodrop is a much simple way. As Siddharth said u need to be careful while procuring nanodrop .
Nanodrop spectrophotometer is the right choice. You need just 1 microliter of DNA solution to get a reliable mesure of your DNA concentration. I've used Nanodrop to quantify a huge amount of DNA and cDNA samples, the latter of which utilized to perform quantitative Real-time PCR experiments.
Nanodrop is the best as it just takes 1ul to measure the concentration. Also you don't have to dilute the sample as you do for spectrophotometer. Saves time as it takes 30sec to measure each sample.
of course nanodrop, but need good eyesight because only 1ul is enough for measurement and have to sit on the center of the specific place.
Aditi Banerjee
Use 1.2 µL to load onto the nanodrop to be sure that the machine uses 1 µL for measurement.
Hi ... you can use Nanodrop to quantify the DNA . it will give u the conc. at 260/280 by which u can determine the purity and exact conc. of ur sample.
If you are only looking for DNA concentrate measurement i would prefer you to go for Picogreen methodology. The kit is supplied by invitrogen. Picogreen is a dye which binds only to Double strand DNA (not Single strand) and gives exact concentration upon comparing with a standard graph prepared by using kit lambda DNA. As there are serial dilutions prepared you will get most accurate values. But the product purity cant be measured by nanodrop only
Hi, you may use pellet paint to concentrate the DNA after lysing the cells, you can check my page for new lysis buffer (TZ) for the isolation of genomic DNA. there are diffrent methods to measure DNA such as fluorometer and nano-drops
Hi, you can use Nanodrop for finding ot the concentration of DNA.
I work in a biobank and we all use Nanodrop it is very useful to know the purity of your DNA but spectrophotomers gives you nucleotides mesure and even if your DNA is degraded you can obtain very nice result with Nanodrop so the best is Nanodrop quantification plus DNA integrity visualization on an agarose gel. Some people think the is fluorimetry with a commercial kit, for ex , Picogreen that gives you the measure of double strain DNA. For me nanodrop plus agorose gel visualization is the best choice, simple and cheap.
Nanodrop is fine if available other wise gel electrophoresis with lambda DNA marker concentrations is the best for most of the studies
I think that Nanodrop is ok for measure the concentration of DNA , and if it is not sure they have run on gel how they are.
Please guys, stop writing that Nanodrop is the best way to go for measuring DNA as well as RNA concentration. I think everyone got that now. Thanks!
Most easiest is nanodrop, but you can verify Gel with known standards
There is a nice fluorometer for DNA and RNA and protein concentration. Qubit Fluorometer from Invitrogen
http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/Qubit.html
Sure, Nanodrop is the best known device for this purpose, but we decided to buy a Picodrop. Why? Because we work with very small amounts of sample material and with the Picodrop you have a 100% sample recovery.
http://www.picodrop.com/
Nanodrop is nice and easy for DNA concentration BUT....it does not give good results for samples that have RNA contamination. I use both Nanodrop (purefied DNA) and Invitrogen's Qbit (crude genomic preps).
Picodrop is a good choice. you can use 2ul of the DNA and doesnt need to dilute. just put the sampler with the tip in the instrument and after you knew the concentration you will be able to recover the content of tip without contamination. For more information go to link
http://www.picodrop.com/
Good luck
@Heng Seiha---I do genomic DNA preps from fungi all the time. These preps have a very large amount of RNA in them. The same is true for plant and bacterial DNA preps. All living cells have both DNA and RNA in them. You will not get an accurate reading of the DNA content of a sample using a nanodrop from these types of preps. I use Qubit for that. When I gel purify DNA slices from agarose gels, I use the Nanodrop, very accurate for that and faster.
You may use Nanodrop Spectrophotometer (NanoDrop Technologies, USA).
It requires only 1-2 microlitre of samples & give better result.
All the best.
Ashutosh
India
We used to use nanodrop to measure genomic DNA concentration, but concentrations were absolutely overestimated, we were mostly measuring free nucleotides resuted from degradation. We use now qubit which only measures double helix DNA with fluorimetry and the results are much more real.
I am interested how many people have suggested to use NanoDrop to measure DNA? :D
I think it is enough now.
Well, if you need to know your concentration exactly, just combine more approaches. Nanodrop or other as a spectrophotometry candidate and Picogreen or Qubit from Invitrogen as a candidate of fluorimetry.
good luck
Nanodrop only gives reliable results when the purity of DNA is high enough. For example, the ratio of 260/280 and 260/230 are above 1.8 or higher and you should get a clear peak at 260nm. Another limitation is that to get reliable results, the detection limit should be higher than 5 ~ 10 ng/ul. I suggest is to combine fluorometic and NanoDrop methods to get more reliable and accurate results.
we are using multiskan go of thermo scientific.It is a very good device for nucleic acid quantification.It comes with a microdrop plate and 16 samples can be quantified at once.only 2 ul of sample is needed
i would go for flourimetry (e.g. Qubit) , NOT spectrophotometry ( e.g. nanodrop)
Though Nanodrop (spectrophotometer) is useful ,it finally depends on your requirement ,I mean if you want to quantify your DNA for real time applications,NGS then certainly you need more relaible instrument like Qubit, other wise for routine uses Nano drop is OK
Our lab recently purchased a BioDrop TOUCH. It is Europe's alternative to the Nanodrop and it is a VERY nice piece of equipment :o) It is as accurate and reliable as the Nanodrop, much easier and faster to operate and has a LOT more features. I highly recommend it.
me to recommended for Nanodrop . i am also using this instrument for measuring DNA concentration,and purity of DNA.
I will have to vote against the Nanodrop, with a stipulation. We have a DNA synthesis guru who only uses the Nanodrop to quant ssDNA that she has synthesized and run on HPLC or PAGE denaturing gels, AND she does NOT use the ng/uL reading from the Nanodrop, but instead uses only the absorbance and a calculated coefficient for that exact oligo. The Nanodrop is also the easiest/fastest quant method. In my experience, I see about a 2X increase in measured quant with the Nanodrop compared to the Qubit, and I believe the Qubit is correct. I use the Qubit. Another great approach is a bioanalyzer...if you have one or can afford it. The BA is probably the best answer, because you can get a quant within a size range. We have switched from qPCR to the BA for sequencing applications, and it works great.
Your term "appropriate" may be misleading as it depends on what you want to do with your DNA. If you are only concerned with ball park concentrations for standard PCR or PCR/RFLP, then nanodrop/gel visualization are probably the cheapest and quickest approach. If, however, you need very accurate measurements of DNA concentration for more advanced downstream applications (GWAS, etc), then i would defer to method such as fluorimetry. Essentially, the nanodrop, or any spectrophotometer) is a "magnifying glass" whereas the Qubit (or similar) is a "microscope" when it comes to "looking" at DNA concentrations.
As mentioned... we use Nanodrop.
It only uses a small portion of your sample, is fast to use, and is easy to use.
Qubit dsDNA HS or BR kits would be the best option. However measurements can vary from one batch of dye to another. So I wouldn't be very surprised if DNA measurements using Qubit would match exactly the ones measured by Nanodrop.... sometimes!!
Nanodrop spectrometers have been a successfully used over years. However, Qubit (fluorometer) based quantification is more sensitive. This device is equally useful for:
- DNA quantification
- RNA quantification &
- Protein quantification
It really depends on your downstream application. For some applications you would ideally want to use the QuBit system and Nanaodrop. Nanodrop always overestimates DNA concentration but broader wavelength analysis is required to measure purity. QuBit (HS or BR) is very accurate but is only designed to give you a DNA (or RNA) measurement.
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