Protocol for the Preparation of DNA Dilutions for use in the
lab
Introduction
Stock DNA is routinely prepared from whole blood by the Nucleic Acid Extraction
Centre. Occasionally it may be necessary for us to extract the DNA ourselves eg. when
blood is received from Dublin. Before use in the lab, the stock DNA is quantitated by the
Picogreen Fluorescent Assay. Once the concentration of the stock DNA is known,
dilutions may be prepared. Stock DNA may be ‘normalised’ to 250ng/ul or 100ng/ul.
DNA is diluted to 5ng/ul for normal lab use.
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.
Materials
TRIS-HCL 1M Solution (Sigma T-2788)
Ethylenediaminetetraacetic acid disodium salt solution (EDTA), 0.5M (Sigma E-7889)
Suitable tubes : For normalisation use Sarstedt 0.5ml tubes – Part Number 72.730.100
For dilution to 5ng/ul use Sarstedt 2.0ml tubes – Part Number 72.694.100
or Treffclear 1.5ml tubes – Part Number 96.7811.9.05
Gilson P1000 pipette and D1000 tips (Anachem)
Gilson P200 pipette and D200 tips (Anachem)
Gilson P20 pipette (Anachem)
Microsol 3+ (Anachem)
White Tissue from roll
Microcentrifuge - Heraeus Biofuge Pico
Vortex
Stock DNA of known concentration
Procedure
1. Calculate the amount of TE0.1 and stock DNA to be added. For example, the
following equation may be used:
Required final conc of DNA(ng/μl) x Final Vol (μl) = Vol of Stock to add (ul)
Actual conc of stock DNA (ng/μl)
Worked Example:
1ml(1000ul) of dilution is required at a final concentration of 5ng/ul.
The stock DNA is at an actual concentration of 500ng/ul.
5 ng/μl x 1000μl = 10ul
500 ng/μl
10ul of Stock DNA is added to 990ul of TE0.1 to make 1ml of dilution at 5ng/ul.
2. Check that there is sufficient TE0.1 to prepare all the required dilutions. If
necessary, more may be prepared as follows (instructions are for the preparation
of 500mls. The volumes may be doubled to prepare 1 litre:The concentrations of the stock solutions are 1M Tris and 0.5M EDTA.
The final concentration of TE0.1 is 10mM Tris and 0.1mM EDTA.
 Thoroughly rinse a 500ml volumetric flask and 500ml Schott bottle (used
only for the preparation of TE0.1) at least 6 times with MilliQ 18.2MΩ
water. Label with the solution name, date and your name.
 Using Gilson pipettes add the correct amounts of Tris and EDTA to the
volumetric flask as follows: The volume of Tris to add is 10mM/1000mM x 500mls = 5ml.
 The volume of EDTA to add is 0.1mM/500mM x 500mls = 0.1ml (100μl).
 Make the volume up to the 500ml mark using MilliQ 18.2MΩ water.
 Seal with the bung and holding over the sink, invert several times to mix.
Transfer into the 500ml Schott bottle.
 Attach the Schott bottle lid loosely, and leave over to the Media labs for
autoclaving.
 Allow the solution to cool to room temperature before use.
3. Decontaminate the work area spill tray with Microsol 3+. Refer to the protocol for
‘Decontamination using Microsol 3+’.
4. Lift the DNA Stock tubes out of the fridge. Vortex briefly and pulse spin in the
microcentrifuge for 5 seconds. Place the tubes, in order, into a 96-well tube rack
at positions B1 – B12 as in Figure 1.
5. Place empty 0.5ml, 1.5ml or 2ml tubes into positions H1 – H12 in the tube rack as
in Figure 1. Copy the lab number from the DNA stock tubes onto the lid of the
opposite empty tube, writing the concentration that the dilution will be prepared to
on the lip of each lid.
6. Double check that the correct dilution tube is assigned to the right DNA stock.
7. Using a Gilson P1000 and D1000 tip transfer the required amount of TE0.1 into
each of the dilution tubes in H1 – H12, moving each tube back one position as the
TE0.1 is added. The tubes are now in positions G1 – G12 as in Figure 1.
8. Using a Gilson P200 or P20 (depending on volume to be transferred) and D200
tips, transfer the required amount of DNA into each dilution tube, taking a clean
tip for each sample, and moving both the stock tube and the dilution tube back by
one position as the DNA is added. The stock tubes are now in positions A1 - A12
and the dilution tubes are now in positions F1 – F12 in the tube rack as in Figure
1.
1
2
3
4
5
6
7
8
9
10
11
12
A
B
C
D
E
F
G
H
KEY
= Position of Stock DNA after dilution has been made
= Position of Stock DNA before dilution is made
= Position of 1.5ml tubes containing TE 0.1 + DNA
= Position of 1.5ml tubes containing TE 0.1
= Position of empty 1.5ml tubes
Figure 1 – Showing how to lay out the tube rack during preparation of DNA dilutions.
9. Vortex the new dilutions briefly and pulse spin in the microcentrifuge for 5
seconds.
10. Leave in the fridge overnight to ensure that the solution is homogeneous.