Introduction: Using the Fluorometer to Quantify DsDNA

The fluorometer allows accurate quantification of dsDNA. This protocol will make the quantification process run smoothly in Dr. Richard Robison's lab. Protocol has been changed based off the Quant-iT Picogreen assay protocol. The TBS-380 (Fig. 1) functions the same as the QuantiFluor-ST system.

We have two different adapters that can be used for the fluorometer; Minicell (Fig. 2) or the PCR tube (Fig. 3). The Robison lab uses the PCR tube adapter with 0.5 PCR tubes (Axygen . The adapter is multidirectional and can be inserted into the optical kit sample compartment in any orientation. The minimum volume required is 100ul.

If the lab runs out of the PCR tubes, you can also use the Minicell adapter found

Step 1: Materials

Reagents Located in DNA Freezer
1. DNA standards
           a. One tube from the 2.0ug/mL box
           b. One tube from the 0.2ug/mL box
2. Picogreen box

WARNING: Picogreen is light sensitive; make sure to minimize exposure to light.

Items Located in Molecular Hood
1. Micro-centrifuge rack (x2)
2. 600ul sterilized eppendorf tubes
3. 100ul glass cuvettes
4. 50ml sterilized 1X TE buffer
5. 10ul; 100ul; & 1000ul reagent pipettes
6.  2;10ul; &100ul DNA pipettes
7. 10ul, 100ul, &1000ul pipette tips
8. Thin black sharpie marker
9. Small container labeled "Pico"
10. Waste container

Items Located Outside of Molecular Hood
1. Vortex
2. Centrifuge
3. Nitrile or Latex gloves (x2)
4. DNA samples to be quantified
5. Fluorometer lab notebook & pen
6. Fluorometer
7. Computer with flurometer excel sheet

Step 2: Filling Out the Fluorometer Notebook

1. The fluorometer notebook has a table of contents behind the front cover
          a. write the page number and title your work
2. Flip to the page that was just numbered and write a brief summary of what will be done for this run and write the date

Make note of any discrepancies outside of this instructable procedure

Step 3: Reagent Preparation

1. With gloves on, take one tube from both the 2.0 & 0.2ug/mL DNA standards box
2. Determine how many tubes you need from the Picogreen box
          a. Count how many DNA isolates that need to be quantified and divide by 42; always round your answer up

Example : If working with 50 samples, duplicates or triplicates will be used for each isolate. For triplicates, 50*3=150/42=3.57≈4 picogreen tubes

          b. Picogreen is also needed for the DNA standards. These standards have a known DNA concentration so they will be 
              used to determine the concentration of the DNA samples. 11 tubes are used for standards.

Example : With the 150 samples, add 11 tubes for the standards. Then divide that sum by 42 to determine the final amount  of
                   picogreen needed 150+11/42=4 tubes of Picogreen. Grab 4 tubes of picogreen from the freezer.

3. Place your reagents on the micro-centrifuge rack located inside the molecular hood
          a. Place DNA standards off to the far left side of rack

4. Place the Picogreen tubes in the small container labeled "Pico" to protect them from light (figure 1) or put it on the picorack in the drawer by the the molecular hood. As long as it is stored in a dark place, it will be fine.

Step 4: Set Up

50 DNA samples will be used as an example for this protocol

1. Place fifty 600ul eppendorf tubes, for your DNA samples, on the micro-centrifuge rack as indicated in figure 1. Place your duplicate/triplicate tube right behind the first tube.

2. Place twelve 600ul eppendorf tubes towards the bottom of the DNA samples, for your DNA dilution standards, on the micro-centrifuge rack
          a. With a sharpie marker, label the twelve bottom tubes 1-11 and circle the numbers (figure 2)

Step 5: Creating DNA Dilution Standards

1. Using the 100ul reagent pipette, add the corresponding 1x TE buffer amounts to the labeled tubes (figure 1)

           Example: tube 12 receives 100ul of 1x TE buffer

Make sure the standard DNA tubes are fully thawed before moving to step 2

Make sure you add the correct DNA standard concentrations to each tube

2. Using the 10ul or 100ul DNA pipette, add the corresponding DNA standard amounts to the five labeled tubes (figure 1)

          Example: tube 4 receives 1ul of the 0.2ng/mL DNA standard

3. Write the standards that are used in the fluorometer notebook including the DNA standard concentration

Step 6: Assay Set Up


Prepare the standards by aliquoting the lowest DNA concentration first; to prevent any contamination. Start with standard 11 and then close the cap.

1      25ul   2.0ng /ul standard + 75ul 1X TE
2      20ul   2.0ng/ul standard + 80ul 1X TE
3      15ul   2.0ng/ul standard + 85ul 1X TE
4      10ul   2.0ng/ul standard + 90ul 1X TE
5        5ul   2.0ng/ul standard + 95ul 1X TE
6        3ul   2.0ng/ul standard + 97ul 1X TE
7        1ul   2.0ng/ul standard + 99ul 1X TE
8     0.5ul   2.0ng/ul standard + 99.5ul 1X TE
9        1ul   0.2 ng/ul standard + 99ul 1X TE
10   0.5ul   0.2ng/ul standard + 99.5ul 1X TE
11    100ul 1X TE

Sample set up

The highest concentration the flurometer can read is 0.2ug. If the DNA is known to be higher than 0.2ug, perform the necessary dilutions to get a DNA concentration less than 0.2ug. Always perform at least a 1:20 dilution of the DNA samples . If you're working with heavily concentrated DNA (500ng/ul), a 1:200 dilution should be fine.

1. Add 95ul of 1X TE buffer to all the sample tubes (including it's duplicate/triplicate tube).

2. Add 5ul of the diluted DNA sample to the 95ul 1X TE tubes. Dispense pipette tube after each aliquot of DNA (figure 1). There should be a total volume of 100ul at this point.

3. Turn off the molecular hood light

4. Grab the picogreen

6. Using the 1000ul reagent pipette, add 8520ul of 1x TE buffer in each Picogreen tube

7. Briefly vortex all Picogreen tubes to mix

8. If possible, centrifuge the picogreen tubes

9. Aliquot 100ul of Picogreen (with a new pipette tip for each aliquot) to all 161 tubes (figure 2) starting with the lowest standard (100ul 1X TE). Cap the tubes after every picogreen aliquot.
          a. the Picogreen tube should contain enough Picogreen for 42 samples; empty the tube out.

10. Grab another Picogreen tube and repeat step 9 until all the DNA samples including the standards have received 100ul of picogreen

Step 7: Fluorometer Set Up

1. Place cuvettes on the second micro-centrifuge rack in the same layout as the eppendorf rack (figure 1)

2. Close all the caps on the eppendorf sample tubes and label them 1-100

3. Briefly vortex all the tubes

4. After all tubes have been vortexed, incubate the tubes at room temperature for at least two minutes

5. Aliquot 60ul from sample tube 1 into cuvette 1. The fluorometer can read a minimum of 50ul. As long as you have more than 50, it's good

6. Repeat step 5 for the remaining tubes to their corresponding cuvette

        Example: 60ul of DNA isolate 10 is transferred to the cuvette 10

Step 8: Fluorometer Readings of DNA Standards

Before reading each cuvette, make sure there are no bubbles in them. The bubbles will affect the accuracy of the reading.To remove the bubbles, gently flick the side of the cuvette with your fingers until they pop.

1. Take the cuvette rack to the fluorometer
2. Turn on the fluorometer
3. Open the lid and place standard dilution tube 1 in fluorometer (figure 1)
          a. The fluorometer should be on the blue default setting
4. Close the lid and press read
5. Write down the value, that appears on the screen, in your lab notebook (figure 2)
6. Discard the read glass cuvette in the glass waste container (located to the left of the fluorometer)
7. Read and record the remaining 11 standard dilutions

Step 9: Fluorometer Microsoft Excel Sheet

1. On the computer desktop to the right of the fluorometer, click on the Microsoft Excel sheet titled Fluorometer

2. On the Excel sheet, insert the fluorometer value for standard dilution 1 (tube 1) under the "0" column/cell (figure 1). This should be the lowest fluorometer value.

3. Enter the remaining 11 standard fluorometer values into the correct column as noted in figure 1. There should be a decrease in flurometer values as you move down the row. If one of the isolates doesn't follow this pattern, it would be best to remove that point from the other 10 standards.

4. Once all 11 standards are entered, a linear regression equation and R^2 will appear (figure 2)
          a. To double check that the points on the line correspond to the data in the cells, click one of the points on the graph. A selection
          grid will appear on the some of the cells. Cells A8-J8 (DO NOT INCLUDE K8 ) should be selected.
          b. Any r^2 value below 0.99 is unacceptable and standards must be remade (go back to Creating DNA Dilution Standards )
          c. Record the r^2 value and equation of the line in the fluorometer notebook

An important thing to look at is if the points looks oversaturated (figure 3). The last point is starting to show a slight curvature indicating oversaturation. This may happen due to dramatic increases in DNA standard from one standard to the next (from 20ul then to 50ul). This usually will not be a problem as long as there are more gradual increases in DNA stnadards (20ul; 40ul; then 50ul).

Even with these precautions, over saturation may still occur. Usually to fix the saturation, removing the point causing the curve will fix it (figure 4). In this case it was the highest point.

Step 10: Fluorometer Readings of Samples & Data Analysis

Before reading each cuvette, make sure there are no bubbles in them. The bubbles will affect the accuracy of the reading. To remove the bubbles, gently flick the side of the cuvette with your fingers until they pop.

1. If the R^2 value is acceptable, read the 50 samples (including their duplicates) on the fluorometer and record the value in the lab notebook

       Example:  Microorganisms species: {78301
                                                            duplicate reading on the bottom

The duplicate reading should be a similar value to the first reading. If not, there is enough sample in both the tubes to reread it. If one of the tubes has a fluorometer reading near the 11th's standards tube value, that probably means no DNA was added. Remove that fluorometer value if that is the case and rerun that sample.

2. Before entering the fluorometer values in the excel sheet, review the range of the fluorometer values for the 1:20 diluted samples.
          Make sure that none of the sample readings are higher than the highest standard value. If that happens you'll have to rerun that  
          sample at a higher dilution.

3 . Enter in each of the individual values that you read into the first and second blue cells as indicated in figure 3. The number in the purple cell indicates the concentration of dsDNA in ng/ul, but this maybe be incorrect.

4. To check that the concentration is correct, double click on the purple cell and calculations should appear as seen in figure 4
          -They should use cells A-J, cell K is not included in both the graph and the purple calculation cell.
          -The red arrow pointing towards the 10 in figure 2 represents the amount of DNA that was aliquoted
                   *If 5ul of DNA was aliquoted this 10 must be changed to 5 (also assuming that 95ul of 1X TE buffer was added)

    Remember that these samples had a 1:20 dilution so look at the 1:20 dilution cell on the excel sheet. It's the purple cell multiplied by 
    20. That is the value that needs to be recorded.

5. Once these calculations are correct, record the DNA concentration, that appeared in the purple cell, in the lab notebook

6 . Record the DNA concentrations on each DNA tube with a thin sharpie marker (figure 3)

Step 11: Cleaning Up

1. If not done already, discard all used glass cuvettes in the glass disposal box
2. Discard all used eppendorf tubes in the waste container located in front of the fluorometer (figure 2)
3. Turn off the fluorometer
4. Put the micro-centrifuge rack in the molecular hood
5. Put away any of unused Picogreen tubes in the molecular reagent fridge to be reused again
6. Place the 2.0 & 0.2ng/mL standards back in the DNA freezer
7. Place the your DNA samples in the 4°C fridge
8. Close the hood and turn on the timed UV light switch