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  • qubit/picogreen

    I have a question about the qubit assay kit for measuring concentrations of dsDNA. The kit comes with 2 standards as tubes. The standards are labeled as 0ng/ul and 10ng/ul.

    I calibrate the qubit with these standards just fine. First I make up the Picogreen mix, then take 10ul of each standard and dilute into 190ul of Picogreen mix, so the standards are diluted 1/20. After qubit is calibrated, I measure the standards in the kit, they read as <0.50ng/ml (for the 0ng/ul tube) and >600ng/ml (for 10ng/ul tube).

    My question is how does the qubit calculate the 10ng/ul labeled standard tube as being greater than 600ng/ml when it is diluted 20-fold in the first place? Can someone show me the calculation of how it does this? I only want to know how it reads the standards as it does.

  • #2
    when I used Qubit HS (high senstivity kit) for measuring gDNA conc., I got entirely different concentation compared to BR (broad range) kit. do you know, what is the difference between Qubit HS kit and Qubit BR kit for DNA conc.?

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    • #3
      Well, from the Life Tech website...

      The BR kit has a range of 100pg/uL to 1000ng/uL and the HS has a range of 10pg/uL to 100ng/uL. If you're on the extreme of the BR kit (Sample is 0.150 ish ng/uL), chances are the assay isn't going to read very accurately compared to the HS kit where you're further away from the lower bound. Same goes for HS if you're at the upper bound, which would place you comfortably in the middle of the BR kit.

      We usually use the HS kit for PCR product and the BR kit for gDNA, as our yields for each usually sit in the middle of each kit's range.

      Originally posted by mmmm View Post
      when I used Qubit HS (high senstivity kit) for measuring gDNA conc., I got entirely different concentation compared to BR (broad range) kit. do you know, what is the difference between Qubit HS kit and Qubit BR kit for DNA conc.?

      Comment


      • #4
        The concentration values that are shown on the Qubit are the concentration of DNA in the tube that you are reading, and not of the original sample. This means that you need to take the value that it gives you in ng/ml and convert it to the ng/ul of your actual sample.

        I don't remember the exact equation to do that off the top of my head, but it's given in the Qubit manual so I'd suggest you take a look at that. I can say that when using the HS DNA kit, if we use 2ul of sample, then the raw value that is given by the Qubit in ng/ml can be converted to ng/ul for the sample by just shifting the decimal point to the left (e.g. if the Qubit says the conc. is 50ng/ml, then the actual conc. of the sample is 5ng/ul).

        This is the biggest issue we have with new people using the Qubit, they forget to convert the raw values to the actual conc. of the sample and that can have very bad downstream effects on library preparation.

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        • #5
          The instrument also lets you "calculate stock concentration" for each sample. Just click the "Calculate Stock Concentration" button after reading a sample, set the units to ng/uL (Or whatever else you want it to be), and set the amount in uL you put into the sample tube (Usually 2uL on our end). It will calculate the actual concentration of sample, after which you can click "save" and it will record the actual concentration onto the local memory.

          You have to do this for each sample, which is a little annoying (Clicking save each time, it remembers the amount in uL and units desired), but it moves pretty quickly.

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