Quantify DNA concentration using Qubit_24773641

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title: "TheNessLab : Quantify DNA concentration using Qubit"

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TheNessLab : Quantify DNA concentration using Qubit

Created by Wen Chen, last modified on Oct 30, 2018

Use this protocol to quantify DNA concentration before performing PCR.

Materials:

• Qubit^TM dsDNA BR buffer
• Qubit^TM dsDNA BR reagent 200x concentrate in DMSO
• Qubit^TM dsDNA BR standard #1
• Qubit^TM dsDNA BR standard #2
• Qubit^TM dsDNA assay tubes
• Qubit^TM fluorometer
  

Step-by-step guide

1. Take the standards out of 4C refrigerator.

2. Prepare working solution (WS) by mixing the reagent and buffer using:
   ***               x*** * 1 uL reagent + x * 199 uL buffer where the total volume of WS required=x*200
                  where x = number of DNA samples you will analyze + 2 standards

3. Prepare standards #1 and #2 in two separate tubes using: 
                  10 uL standard # + 190 uL WS

4. Prepare sample tubes using: 
   ***               y*** uL DNA sample + 200-y uL WS, where 1 uL≤y≤20 uL depending on the estimated DNA concentration

5. Start Qubit by plugging it in and touch the screen. 

6. Select "dsDNA"→"dsDNA: Broad Range"→ "Read standards."

7. Open Qubit lid. Wipe the standard #1 tube with Chemwipe. Place tube in Qubit. Close the lid.

8. Select "Read standard." Remove tube when the read is complete.

9. Repeat Steps 7 and 8 with standard #2 tube when prompted to insert standard #2.

10. Select "Run samples" and repeat steps 7 and 8 with sample tubes.

Tips:

• Allow assay tubes to incubate for at least 2 minutes before reading.
• Fluorescence signals are stable for up to 3 hours at room temperature.
• Perform the readings when the assay tubes are at room temperature. This is especially important when you are performing multiple readings per tube because readings can increase the tube temperature and temporarily reduce fluorescence.
• Increase y if you think your DNA samples have low concentrations and vice versa.

Related articles

Qubit 3.0 Fluorometer manual

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