DNA Purification from Blood or Body Fluids
OBJECTIVE
To isolate DNA from total blood using the Qiamp DNA Blood Mini kit (51104 Qiagen)
SAMPLE
400 mcL of total blood from a frozen EDTA tube. Patient MªRosa Soldati (CNTN1-HLA study patient from Milan, number 16)
PROTOCOL (DNA purification from blood or body fluids, spin protocol)
This protocol is for purification of total (genomic, mitochondrial, and viral) DNA from whole blood, plasma, serum, buffy coat, lymphocytes, and body fluids using a microcentrifuge.
Things to do before starting
■ Equilibrate samples to room temperature (15–25°C).
■ Heat a water bath or heating block to 56°C for use in step 4.
■ Equilibrate Buffer AE or distilled water to room temperature.
■ Ensure that Buffer AW1, Buffer AW2, and QIAGEN Protease have been prepared according to manufacturer’s instructions
■ If a precipitate has formed in Buffer AL, dissolve by incubating at 56°C.
Procedure
1. Pipet 20 μl QIAGEN Protease into the bottom of a 1.5 ml microcentrifuge tube.
2. Add 400 μl sample to the microcentrifuge tube.
3. Add 400 μl Buffer AL to the sample. Mix by pulse-vortexing for 15 s.
4. Incubate at 56°C for 10 min.
5. Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.
6. Add 400 μl ethanol (96–100%) to the sample, and mix again by pulse-vortexing for 15 s.
7. Carefully apply the mixture from step 6 to the QIAamp Mini spin column and centrifuge at 6000 x g 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. Place the QIAamp Mini spin column in a clean 2 ml collection tube (provided), and discard the collection tube containing the filtrate.
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 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. This step helps to eliminate the chance of possible Buffer AW2 carryover.
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. Carefully open the QIAamp Mini spin column and add 100 μl Buffer AE.
Incubate at room temperature for 5 min, and then centrifuge at 6000 x g (8000 rpm) for 1 min.
RESULTS
81.8 ng/mcL. 260/280: 1,81
