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Genspace QuBit Protocol (Archived)
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Genspace QuBit Protocol

Introduction

The Qubit fluorometer is a device that quickly and specifically quantitates DNA or RNA.

Materials

    • Micropipettes and Tips
    • QuBit Standard Tubes
    • Dilution Buffer
    • Eppendorf Tubes
    • QuBit Tubes
    • QuBit Device
    • DNA/RNA Sample

Procedure

  • Securing Reagents and Tubes
  1. Get the two standard tubes from a small, clear zip-lock bag on the door of the non-microbial 4C fridge.  They are labeled “1” and “2” on the top with marker.  (These are the 0ng/uL and 100ng/uL references.)  Briefly vortex these for best results.
  1. Locate the dilution buffer (large bottle) among lab supplies against the wall.  The 200x intercalating reagent is in a small tube in the black photosensitive reagent box on the metal shelf.
  1. Under one of the lab benches, you will find a bag labeled “FOR QUBIT ONLY”: make sure to use this.
  • Prepare Master Mix
  1. Suppose you have N samples to analyze.  Put 200*(N+2.5) uL of dilution buffer into an Eppendorf tube (or multiple Eppendorf tubes as needed).  (This is enough for the N samples plus 2 standards plus a little extra to deal with pipette variations.)
  1. Add (N+2.5) uL of the 200x intercalating reagent. Vortex.  This is the master mix.
  1. Example: if you need to analyze 5 samples, N=5 and you will put 1.5mL of the dilution buffer and 7.5uL of the 200x intercalating reagent into the master mix.
  • Prepare Standard Samples
  1. Suppose you have N samples to analyze.  Put 200*(N+2.5) uL of dilution buffer into an Eppendorf tube (or multiple Eppendorf tubes as needed).  (This is enough for the N samples plus 2 standards plus a little extra to deal with pipette variations.)
  1. Add (N+2.5) uL of the 200x intercalating reagent. Vortex.  This is the master mix.
  1. Example: if you need to analyze 5 samples, N=5 and you will put 1.5mL of the dilution buffer and 7.5uL of the 200x intercalating reagent into the master mix.
  • Prepare Test Samples
  1. If you are analyzing PCR results, I recommend adding 1uL of DNA input to 199uL of the master mix.  Vortex.
  1. If you are analyzing plasmid preps, I recommend adding 2uL of DNA input to 198uL of the master mix.  Vortex.  (If the plasmid copy number was small, then you may get “DNA TOO LOW” errors, in which case you may need to add 10uL of DNA to 190uL of master mix.)
  1. Note: the total volume in every tube will always be 200uL.
  1. Incubate all samples (standard and test) for two minutes at room temperature.
  • Find the Concentration Using the QuBit
  1. Hit the GO button on the machine and follow instructions.
  1. You will select “Run a new calibration” and sequentially load S1 and S2 as instructed.
  1. You will then sequentially load each test sample.  After each sample is loaded, you will select getting DNA concentration and scroll with the down button to the amount of DNA you added (1uL, 2uL or other).  The answer is given in uL/mL, but this is, of course, also the same as the more useful ng/uL.

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