I am purifying psPAX2 & VSVG plasmid with invitrogen Megaplasmid kit but the problem is when I am taking the reading for the sample, there is a huge difference. I am taking the reading 4 times for the SAME plasmid (eg. psPAX2), and to my surprise the difference is huge in ng/ul. eg for psPAX2
Reading 1. 434 ng/ul,
Reading 2. 400 ng/ul,
Reading 3. 354 ng/ul,
Reading 4. 405 ng/ul,
Although the ratio for High quality DNA: A260/A280=1.8~2.0; A260/A230>2 remains more or less the same & in some repeats, exactly the same. For me this means it has no RNA/DNA/Phenol contamination. In my case ALWAYS A260/A280> 1.8 (1.87-1.9) & A260/A230>2.2 (2.29-2.34)
Please suggest what is the problem, and what concentration I should take (for eg from the above 4 readings of psPAX2 plasmid conc.) for my lentivirus preparation.
Please kindly suggest. Many thanks.
NOTE: I have taken the reading on two different nanodrops but the difference were more or less the same. Also I tried Zymo plasmid miniprep kit again their were differences in the reading of the same sample/plasmid.
Actually nanodrop is not a quite accurate method especially when trying to measure plasmid concentration. Your readings look like oscillating around -400 ng/ul, which I would take.
Hello Syed,
I have also found the Nanodrop readings to be highly variable, so I only use it when I have low sample concentration, or a just need a quick estimate of how much of a particular sample I have. DNA has a 260/280 ratio of 1.8-2.0 in 10mM Tris pH8.5...if you are in any other buffer, or at a different pH this will affect your ratio. You can still have contaminants and get a ratio between 1.8-2.0. Phenol absorbs strongly between 270-275. contaminating particles in your solution or dirty optics will give absorbance around 325nm. I suspect your sample is actually ok however, so if you have access to an actual spectrophotometer and quartz cuvettes, I would use that instead if you need the accuracy! Just make sure you dilute your DNA sample into 10mM Tris pH 8.5 before your measuring. With your concentration you are reporting above you would only need to use 2-5ul in 1000ul for spec. Otherwise, just keep in mind if you are using the nanodrop that you are only getting a rough estimate of your sample concentration.
Cheers
Ron
Hi Ron,
Many thanks for your kind reply. My DNA is dissolved in TE buffer supplied with the Plasmid purification kit by invitrogen.Thanks for your input I will try tomorrow in 10mM Tris pH8.5, as suggested. Also worthy to mention since I had a doubt that the ethanol (70%) (took in the last step of precipitation of DNA) may interfere with reading, so I did triplet reading of ethanol at the DNAs absorption but found a negative value for the same, so I think ethanol contamination should not be a matter in my case. And sorry I forgot to mention above, the reading which I took was of the sample which were 4 times diluted so actually it is somewhat around 1.2ug/ul, as I am using Megaprep kit.
Anyway, tomorrow I will try with Tris.
Mnay thanks
Syed
Are the 4 different readings from the same dilution, or four separate dilutions of the same plasmid prep? Could it be a pipetting issue?
Nanodrops are notoriously inaccurate (they usually over-estimate the concentration compared to fluorometric methods) but I've not experienced that great of a range in readings from one prep. Maybe 20-30 ng/uL variation but not 80. You could also try reading your blank to make sure the instrument is staying calibrated.
Hi Nicholas,
They are from the same dilutions. This is what surprising for me too. its a bog difference, although with all no variation in 260/280 & 260/230 ratio. But above Ron has suggested to do with 10mM Tris. Tomorrow I will try that. I have already done it with blank and also I did the same readings at an another common floor nanodrop. That was worst than these readings.
Syed
Plz try in the spectrophotometer and confirm the concentration. Nanodrop does give inaccurate readings and I too experienced many times. So, i always rely on spec which always is best.
I agree with Dooyoung. Nanodrop just isn´t very accurate, but at least at such high concentrations it works and you can rest asure that you actually have a lot of DNA, somewhere around 400 ng/ul, which I guess is accurate enough for most applications? If you need an accurate reading, you have to use another platform (qPCR / qubit / picogreen).
Cheers,
Vegard
One of my professors once exclaimed: "This Nanodrop machine is a nano-mistake!" Then he paused, to think about that for a moment. He corrected himself: "No wait, a giga-mistake!".
Hi Syed,
Using the 10mM Tris will only help you to see your sample purity via the 260/280 ratio (and in a spectrophotometer it will help give you a more accurate reading because the constants that you use to calculate concentration are derived from readings taken in 10mM Tris pH 8.5). The variability you are seeing is definitely due to the Nanodrop in this case, so it is not going to help with the between sample variability you are getting. You have not really said why you need to be so accurate, but no matter what you do, you are not going to get the accuracy out of the Nanodrop...If you really need accuracy you will have to switch to a spectrophotometer, or better yet a fluorometer.
Goood luck
Ron
Nanodrop spectrophotometer works pretty well. Clean it carefully, and service now and then. To obtain somewhat more accurate and specific readings (but it takes longer + you need to buy reagents)- use Qubit fluorometer
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