Protocol for the Quantitation of DNA using the Picogreen Fluorescent
Assay
Introduction
Picogreen® reagent is a proprietary, asymmetrical cyanine dye which exhibits a >1,000
fold increase in fluorescence when bound to ds DNA. Picogreen is 10,000 fold more
sensitive than UV absorbance methods and is highly selective for ds DNA over ss DNA
& RNA (Invader/Molecular Probes literature). All stock DNA used by the Nephrology
Research Group is quantitated using the picogreen fluorescent assay before the
preparation of dilutions for use in the lab.
NB: Picogreen is light sensitive → cover all tubes with foil
Plot ‘new’ standards for each plate to account for differences between plates
Safety Notes
Read and sign the COSHH Risk Assessment and record sheet, the Task Risk Assessment
and record sheet and the SOP and record sheet before beginning the procedure.
Labcoat, safety glasses and disposable nitrile gloves should be worn.
DNA is not generally thought to constitute a health hazard, however the DNA has not
been screened to ensure that it is free from viruses therefore good microbiological
practice should be followed to minimise the risk
Take regular breaks when pipetting to avoid repetitive strain injury.
The laboratory Rules must be adhered to at all times
NB: **Picogreen is not classified as hazardous but due to it’s ability to bind to DNA, it
should be handled as a potential mutagen**. Ensure that all picogreen waste is disposed
into cytotoxic sharps box with purple lid.
Materials
Cytofluor Multi-Well Plate Reader, Series 4000, with correct filters installed
Heraeus Biofuge Pico Microcentrifuge
Sorvall Legend MACH 1.6 plate spinning centrifuge
Vortex
Corning Costar 96-well polycarbonate plates (D&H, Part No 6511, Model M)
Adhesive plate seals (AB-0580)
Treffclear 1.5ml tubes (Anachem, Part No 96.7811.9.05), autoclaved
96-place tube rack for 1.5ml tubes
TE Buffer (prepared from 100x solution, Sigma, Part No T-9285), filter sterilised
Calf Thymus DNA (Sigma, Part No D-3664)
Picogreen Reagent, Molecular Probes (Invitrogen, Part No P7581)
Anachem Octapipette 5-50μl
Gilson Pipettes P1000, P200, P20 and Gilson Diamond tips D1000 and D200
Gilson Repeater pipette and distritip Mini
Procedure
Step 1 – Preparation of Calf Thymus DNA Stock
NB. Calf thymus DNA stocks are normally prepared in advance and frozen until needed.
Preparation of stock @ 1mg/ml
1. Prepare the calf thymus DNA stock solution to 1mg/ml by adding 1ml of TE
buffer to the supplied vial containing 1mg of calf thymus DNA powder.
2. Swirl gently for a few minutes until all the powder has gone into solution. Vortex
briefly to ensure thorough mixing.
3. In a tube rack, label the lids of ten 1.5ml tubes with ‘CT . Label the lip of each
tube with the concentration ‘1mg/ml’.
4. Carefully remove the vial seal and bung, and using a Gilson P20 pipette aliquot
the calf thymus DNA stock into the labelled 1.5ml tubes in 100μl amounts.
5. Place the stock in a labelled box in the -20°C freezer for future use.
Preparation of Stock @ 3200ng/ml
6. Remove one aliquot of calf thymus DNA 1mg/ml stock from the freezer and
allow to defrost on the bench. Vortex briefly and pulse spin in the microcentrifuge
for 5 seconds.
7. In a tube rack, label the lids of 25 1.5ml tubes with ‘CT’. Label the lip of each lid
with the concentration ‘3200ng/ml’.
8. Using a Gilson P1000 pipette, add 1246μl of TE buffer to each 1.5ml tube.
9. Using a Gilson P20 pipette, add 4ul of Calf thymus stock DNA to each 1.5 ml
tube.
10. Vortex briefly and pulse spin in the microcentrifuge for 5 seconds.
11. Place the 3200ng/ml stock into a labelled box in the -20°C freezer for future use.
Step 2 – Dilution of the stock DNA for quantitation
Please Note: You need to ‘assume’ an estimated concentration the stock DNA, so that
the [DNA] will fit onto the standard curve. The 1 in 1000 dilution factor outlined below
assumes that the stock is at 1ug/ul. For very dilute stock (eg <100ng/ul) a 1 in 100
dilution factor may be used and for very concentrated stock (eg 2ug/ul or higher) a 1 in
2000 or 1 in 5000 dilution factor should be used ** There are templates available on the
Cytofluor computer to read results at each of these dilution factors – be sure to pick the
correct template to read your results**
Day 1 - Prepare in duplicate a 1 in 100 dilution of the stock DNA
12. Vortex the stock sample and pulse spin in the microcentrifuge for 5 seconds.
13. Label two 1.5ml tubes with the lab number of the stock sample, and the dilution
factor of 1 in 100. Label tube 2 as the duplicate (put a black mark on the hinge).
14. Using a Gilson P200 pipette, add 198μl of TE buffer to each 1.5ml tube.
15. Using a Gilson P20 pipette, add 2μl of DNA stock to each tube.
16. Vortex briefly and pulse spin for 5 seconds in the microcentrifuge.
17. Place in the fridge overnight.
18. NB – Remember to make up at least one control sample of known concentration
for inclusion on each plate.
Day 2 – Prepare in duplicate a 1 in 1000 dilution of the stock DNA
19. Remove the 1 in 100 dilution tubes from the fridge and vortex briefly. Pulse spin
in the microcentrifuge for 5 seconds.
20. Label two 1.5ml tubes with the lab number of the DNA sample and the dilution
factor of 1 in 1000. Label tube 2 as the duplicate.
21. Using a Gilson P200 pipette, add 90μl of TE buffer to each 1.5ml tube.
22. Using a Gilson P20 pipette, transfer 10μl from each of the 1 in 100 dilutions
(prepared on Day 1) into the corresponding 1 in 1000 dilution tube.
23. Vortex briefly and pulse spin for 5 seconds in the microcentrifuge.
24. Place in the fridge overnight.
Day 3 – Preparation of the calf thymus standards and Quantitation of the DNA
25. Remove from the freezer one tube of calf thymus DNA stock @ 3200ng/ml.
Allow to thaw on the bench. Vortex briefly and pulse spin in the microcentrifuge
for 5 seconds.
26. Number two sets of 1.5ml tubes from 1 - 9 and create duplicate (A and B)
sequential dilutions of calf thymus DNA for the standards, as shown in Table 1.
Tube
Number
*CT 3200
1
2
3
4
5
6
7
8
9
Volume of Standard
DNA stock (ul)
4 of stock
600 of CT 3200
600 of 1
600 of 2
600 of 3
600 of 4
600 of 5
600 of 6
600 of 7
0
Volume of
TE (μl)
1246
600
600
600
600
600
600
600
600
600
Tube[DNA] Final Concentration (ng/ml)
(ng/ml)
after Picogreen is added in assay
3200
1600
800
800
400
400
200
200
100
100
50
50
25
25
12.5
12.5
6.25
0
Blank for DNA and Picogreen
Table 1 – Showing how to prepare the calf thymus DNA standards. Each tube numbered
1-9 is prepared in duplicate.
 *Note that this is the tube of calf thymus stock @ 3200ng/ml that has
just been removed from the freezer.
 Make sure to leave the first dilution on the bench for at least 20 minutes
before proceeding. Let all other dilutions rest on the bench for 10 minutes
before transferring.
 Vortex and spin the tubes at each stage, as soon as the calf thymus DNA is
added to the TE and also after the tube has rested on the bench for 10
minutes.
27. While tube 5 is resting on the bench for 10 minutes, remove the ‘Day 2’ 1 in 1000
DNA dilutions from the fridge and leave on the bench to allow to come up to
room temperature.
28. As soon as preparation of the Standards is complete, turn on the Cytofluor using
the switch on the back and open up the software on the computer. Turn on the
lamp (needs 10 minutes to warm up).
29. Prepare a plate layout for the samples to be picogreened, placing the first sample
in well A1 and it’s duplicate in B1, the second sample in A2 and it’s duplicate in
B2 etc, as shown in Figure 1. Place the samples and duplicates in order in a tube
rack. Place Calf thymus standard tube 1 in position G1 and it’s duplicate in H1,
tube 2 in G2 and it’s duplicate in H2 etc. Vortex all tubes briefly and pulse spin in
the microcentrifuge for 5 seconds.
A
B
C
D
E
F
G
H
1






1600
1600
2

D

D

D
800
800
3
S
U
S
U
S
U
400
400
4
A
P
A
P
A
P
200
200
5
M
L
M
L
M
L
100
100
6
P
I
P
I
P
I
50
50
7
L
C
L
C
L
C
25
25
8
E
A
E
A
E
A
12.5
12.5
9
S
T
S
T
S
T
0
0
10

E

E

E
11

S

S

S
12






Figure 1 – showing a general plate layout for the picogreen assay. NB → the samples
of unknown concentration, their duplicates and the standards must all be placed
exactly as shown or the analysis template will not will not read the results correctly.
30. Prepare sufficient 1/200 dilution of Picogreen reagent - Remove 1 aliquot of
picogreen stock from the -20°C freezer. Allow to thaw on the bench. Vortex
thoroughly for 10 seconds.
For one full plate, prepare enough for 100 samples:
To a 7 ml bijou bottle, add 4975μl TE buffer + 25 μl picogreen reagent
Wrap bijou tube in foil and vortex thoroughly for 10 seconds.
31. Place a blank Corning Costar 96-well plate into the Cytofluor. Select the plate
type from the drop down box at the top right hand corner. Check that the
following are set correctly:
Excitation:
Emission:
485 / 20 nm
530 / 25 nm
Press the ‘Start’ button to initiate a read. Save this blank plate read to file
C/cytofluor. Export the .mfr file generated to D/AJ/Picogreen.
32. Remove the plate from the Cytofluor and using a Gilson P200 pipette, add 50μl of
each unknown sample, duplicate and standard according to the plate layout.
33. Using a Gilson Repeator pipette add 50ul of picogreen 1 in 200 dilution to each
well in use **Care should be taken to avoid splashing**. Mix by pipetting up and
down 3 times with the octapipette.
34. Cover the plate with an adhesive seal (AB-0580). Pulse spin in the Sorvall Legend
MACH 1.6 centrifuge with a suitable balance, to a speed of 1500rpm, to remove
air bubbles. Carefully remove the plate seal ** Take care to avoid splashing**,
and read on the Cytofluor within 5 minutes of adding Picogreen.
Refer to ‘Protocol for Picogreen Analysis on the Cytofluor’ for detail of how to save
the results and analyse them using the MS Excel template.
35. The MS Excel template generates a least means squared linear regression analysis
with coefficient of determination (R2). R2 is a measure of the consistency of
prepared standard dilutions. The higher this value, then the more confidence may
be placed in the accuracy of estimated DNA concentrations. Check correlation of
standard curve → Only use if R2 > 0.995. If R2 ≤ 0.995, then repeat the plate
and check volume of TE, dilution factors etc. Check coefficient of variance (CV)
between the samples. Note that calf thymus DNA standards consistently produce
calibration curves with average R2 = 0.9998
36. The template should automatically generate DNA concentrations on the summary
worksheet, both with and without the background plate values. Individual samples
that have failed will be highlighted in red. Save file with unique name and print
the results. [DNA] should be within ±2% of 100 ng/ul, i.e. 98-102 ng. If [DNA]
is outside this range then discard (put in fridge) dilution. Repeat once from the
1/100 dilution (starting from Day 2) and if it consistently fails then create fresh
dilution from the stock DNA.
37. Check that the fluorescence values of the DNA stock of unknown concentration
fall within the range of the standard curve. A very dilute stock which fails may be
successful if picogreened at a 1/100 dilution. A very concentrated stock may fall
outside the fluorescence range and may need to be diluted to 1/2000 or even
1/5000 to fall within the range. MS Excel templates for these dilution factors are
available. Make sure the correct one is selected for analysis.
38. Factors that may interfere with the assay are high salt ( >0.25mM), and trace
amounts of phenol.