PERTEMUAN ke-10

PRAKTIKUM DIAGNOSTIK MOLEKULER 2

CANDIDA ALBICANS DNA ISOLATION FROM URINE

USING QIAamp DNA-Mini KIT

PREPARATION
1. Stool, plasma, serum, urine, cerebrospinal fluid, and other body fluids often contain
very low numbers of cells or viruses. In these cases, concentrating samples from
up to 3.5 ml to a final volume of 200 μl.
2. All centrifugation steps are carried out at room temperature (15–25°C).
3. Equilibrate samples to room temperature (15–25°C).
4. Heat a water bath or heating block to 56°C.
5. If a precipitate has formed in Buffer AL, dissolve by incubating at 56°C.

Free viral DNA from fluids or suspensions

a. For preparation of DNA from free viral particles in fluids or suspensions (other than
urine) using the Protocol: DNA Purification from Blood or Body Fluid
protocols we recommend the addition of 1 μl of an aqueous solution containing 5–
10 μg of carrier DNA to 200 μl Buffer AL.
b. To ensure binding conditions are optimal, increase the volume of ethanol added at
step 5 from 200 μl to 230 μl.
c. Elution should be in 60 μl Buffer AE.

Free viral DNA from eye, nasal, pharyngeal, or other swabs

Additional reagent required: Phosphate-buffered saline (PBS) containing a common
fungicide.
 PROCEDURE
1. Collect samples and transfer to 2 ml PBS containing a common fungicide and
bactericide. Incubate for 2–3 h at room temperature (15–25°C). Centrifugation
for 10 min at 5000 x g (7500 rpm), wash pellet three times with 1 ml of
phosphate buffer saline (PBS).
2. Concentrate the samples from 2 ml to 200 μl.
3. Pipet 200 μl concentrate into a 1.5 ml microcentrifuge tube. Add 20 μl QIAGEN
Protease, add 200 μl Buffer AL to the sample.
4. Mix by pulse-vortexing for 15 s, incubate at 56°C for 10 min.
5. Add 230 μl cold ethanol (96–100%) to the sample, and mix again by pulse-
vortexing for 15 s.
6. Carefully apply the mixture from step 5 to the QIAamp Mini spin column (in a 2 ml
collection tube) without wetting the rim.
7. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp
Mini spin column in a clean 2 ml collection tube (provided), and discard the tube
containing the filtrate.
8. Carefully open the QIAamp Mini spin column and add 500 μl Buffer AW1 without
wetting the rim. Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 min.
9. Carefully open the QIAamp Mini spin column and add 500 μl Buffer AW2 without
wetting the rim. Close the cap and centrifuge at full speed (20,000 x g;14,000 rpm)
for 3 min.
10.Recommended: Place the QIAamp Mini spin column in a new 2 ml collection tube
(not provided) and discard the old collection tube with the filtrate. Centrifuge at full
speed for 1 min.
11.Place the QIAamp Mini spin column in a clean 1.5 ml microcentrifuge tube (not
provided), and discard the collection tube containing the filtrate.
12.Carefully open the QIAamp Mini spin column and add 100 μl (50+50) Buffer AE
or distilled water.
13.Incubate at room temperature (15–25°C) for 1 min, and then centrifuge at 6000 x g
(8000 rpm) for 1 min.