DNA Isolation and Quantitation
L.M. Ball
Introduction
Deoxyribonucleic acid (DNA) encodes information necessary for cell structure, function and
replication. Techniques for recovering DNA from organisms and for quantitating DNA are
fundamental to molecular biology as well as for many aspects of genetic toxicology. This
laboratory excercise will demonstrate one method for recovery of DNA from bacteria, and one
way of quantitating DNA by spectrophotometry. (Many other techniques exist to accomplish
these objectives). For comparison, you will also recover pre-purified DNA from a standard
solution, and quantitate the efficiency of your recovery.
CAUTION::This lab employs strong acid in the quantitation step. Wear gloves.
Wear your lab coats, and safety glasses if you have them. Safety goggles will
be provided.
Procedures
Extraction of DNA from bacteria: You are provided with a pellet derived by
centrifuging approximately 40 x 109 organisms grown in an overnight culture of
Salmonella typhimurium TA100.
1) Wash to remove traces of growth medium: Pour off supernantant into
a labelled beaker so that it can be autoclaved before disposal. Resuspend the
pellet in about 0.5 mL of cold TE buffer, transfer to a 13 x 100 test tube, rinse original tube with
about 2.5 mL of TE buffer, add rinsings to resuspended pellet, mix by inverting the test-tube
covered with parafilm. Centrifuge 5 min at 3000 rpm to collect cell pellet. Pour off supernatant
(into beaker that will be autoclaved).
2) Lysis of cell walls: Resuspend the cells in 0.8 mL of cold sucrose solution.
Add 0.2 mL of Lysozyme solution. Swirl gently for 5 min in an ice-bath.
Add 0.2 mL of cold 0.5 M EDTA solution. Swirl gently for 5 min in an ice-bath.
Add 0.8 mL of Triton solution, mix well. This should produce a viscous suspension of lysed
cells.
Place the lysate on ice to chill for 5 min.
3) Recovery of DNA: Add 1 mL of H2O to the suspension, then layer 2 to 4 mL of cold
absolute ethanol onto the suspension. DNA will precipitate at the interface, RNA and protein
will also precipitate out.
With a Pasteur pipette gently stir the suspension, in one direction only. DNA fibres will wind
onto the pipette.
Remove from the suspension when no further DNA is being picked up, rinse with distilled water
from wash-bottle, allow to air-dry for a few minutes.
4) Redissolve the DNA in 2 mL of SSC solution.
Dispose of residual lysis suspension into beaker for autoclaving.
Recovery of calf thymus DNA from stock solution: You are provided with 3 mg of calf thymus
DNA, dissolved in 3 mL of SSC solution. Reserve 0.5 mL for assay. Add 0.5 mL NaCl solution
to make the remaining solution 1M in NaCl, then add 5 mL of cold absolute ethanol. Recover
the precipitated DNA by winding onto a Pasteur pipette. Redissolve the DNA in 3 mL of SSC
solution.
Dilution of DNA solutions: It will be necessary to dilute the stock calf thymus DNA solution,
and probably also the solutions of recovered DNA, in order to obtain readings that will be in the
range of linearity of the spectrophotometers. Base your dilutions on the extinction coefficient
given below. Dilute the DNA solutions with SSC. Make at least 3 dilutions that bracket your
anticipated range. Also include a reagent blank - a tube with SSC but no DNA.
Quantitation of DNA: Add 1 mL of diluted DNA solution to 2 mL of diphenylamine reagent,
vortex, heat for 10 min in boiling water. Read the absorbance of the resulting blue solution at
600 nm (remembering to adjust dark current and zero with distilled water as before). Calculate
the amount of DNA in your extracts using the extinction coefficient: 0.019/:g/mL/cm path
length.
[Reference: Schneider, Determination of nucleic acids in tissues by pentose analysis. In:
Methods in Enzymology, Vol 3, pp. 680-684, 1957].
Write-up
Describe the experiment in your own words.
Assuming that your dilutions of the stock DNA solution were accurate, how valid is the literature
extinction coefficient ?
How efficient is your recovery of DNA from the stock solution ?
Based on this efficiency of recovery, estimate how much DNA there is in one bacterium.
What might you need to do differently to recover DNA from
(a) an animal cell
(b) a plant cell