Robison: Quantifying dsDNA using the PicoGreen Quant-IT Fluorometer
Procedure written by: Woan Lowe
TABLE OF CONTENTS
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.
Two different adapters can be used for the fluorometer; PCR (Fig. 2) or the Minicell tube (Fig. 3).
The Robison lab uses the PCR tube adapter with the thin-wall, clear 0.5mL PCR tubes (Only use Axygen PCR-05-C or the Qubit assay tubes). 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 PCR tubes, the Minicell adapter can be used (found in the drawers underneath the fluorometer). The adapter has a UV setting and a Blue light setting; the blue light setting will be used. To properly orient the adapter, face the "Blue" text towards the user (Fig. 4).
Reagents Located in DNA Fridge
DNA standards a. Large tube of 2.0ug/mL standard b. Cyrovial of the 0.2ug/mL standard
Reagents Located in DNA Freezer Picogreen (15mL aluminum covered centrifuge tubes)
WARNING: Picogreen is light sensitive; make sure to minimize exposure to light by keeping the vials in the Picogreen white bag. Picogreen works most effective at ambient temperatures. As you set up your samples, place the picogreen white bag in the drawer next to the molecular hood to thaw.
Items Located in Molecular Hood
1. Micro-centrifuge rack (x2)
2. 600µl sterilized eppendorf tubes
3. 0.5mL PCR thin walled tubes (PCR adapter) or100ul minicell borosilicate glass cuvettes (Minicell adapter)
4. 50ml sterilized 1X TE buffer
5. 10µl; 100µl; & 1000µl reagent pipettes
6. 2µl;10µl; &100µl DNA pipettes
7. 10µl, 100µl, &1000µl pipette tips
8. Thin black sharpie marker
9. Waste container
Items Located Outside of Molecular Hood
3. Nitrile or Latex gloves (x2)
4. DNA samples to be quantified
5. Fluorometer lab notebook & pen
7. Computer with fluorometer excel sheet
1. With gloves on, take the rack containing the 2.0µg/mL and 0.2 µg/mL standard.
2. Determine how many picogreen tubes you need from the freezer
a. One tube quantifies about 85 samples.
3. Place your reagents on the micro-centrifuge rack located inside the molecular hood
4. If you haven't already, place the picogreen tubes in the white "pico" bag and place in the drawer next to the molecular hood
25 DNA samples will be used as an example for this protocol
1. Turn the molecular hood light back on
2. Place fifty 600µl eppendorf tubes, to dilute the DNA samples, on the micro-centrifuge rack
3. Each sample will be ran in triplicates. Therefore one sample will have three tubes (Fig. 1). Only use the 0.5mL PCR thin wall tubes for this set up (Fig. 1).
3. Set up the eleven standards by placing eleven 0.5mL PCR thin wall tubes in a micro-centrifuge rack towards the bottom or top of the DNA samples (Fig. 1).
Creating DNA Dilution Standards
Two standards are made in this protocol; 2.0µg/µl and 0.2µg/µl. Serial dilutions will be used to create a standard line.
With a sharpie marker, label the eleven standard tubes 1-11 and circle the numbers (Fig. 1)
Filling out the Fluorometer Notebook
In your lab notebook, write the date and write a brief summary of what will be done for this run.
Quantifying Burkholderia isolates
1:20 dilutions (5µl of DNA and 95µl of 1X TE buffer) of stock DNA. 5µl aliquots in triplicates.