Manual QIAgen DNA Extraction
Clemens Scherzer, Bradley Hyman, Charles Jennings
Abstract
This protocol explains the Standard Operating Protocol for manually extracting DNA using Qiagen.
Before start
DNA Q/C GOALS
- Cary Concentration Assay
1. 260/280 = 1.8-2.0
2. Manual Puragene Extraction: 260 µg /mL (65 µg total) of DNA/subject
3. Automated QIAcube Extraction: 125 µg/mL (50 µg total) of DNA/subject
2. .7% Agarose Gel Electrophoresis
1. Human DNA = 23.13 kb with λ DNA-HindIII digest (NEB)
Steps
Manual Qiagen DNA Extraction
Thaw buffy coats or whole bloods by equilibrating to Room temperature.
Heat a heating block to 56°C.
Gather all the necessary reagents/buffers (Buffer AE, Buffer AW1, Buffer AW2, Buffer AL, 100% ethanol, Qiagen Protease).
If a precipitate has formed in Buffer AL, dissolve by incubating in 56°C.
Gather appropriate number of QIAamp Mini spin columns and 2ml collection tubes.
Pipet 20µL into the bottom of a 1.5ml microcentrifuge tube.
Add 200µL to the 1.5ml microcentrifuge tube.
Add 200µL to the sample and mix by pulse-vortexing for 0h 0m 15s at half-speed.
Incubate on 56°C heat block for 0h 10m 0s.
Briefly centrifuge the 1.5ml microcentrifuge tubes to remove drops from the inside of the lid.
Add 200µL to the sample, and mix again by pulse-vortexing for 0h 0m 15s. After mixing, briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.
Carefully add the sample from step 6 to the QIAamp Mini spin column (in a 2ml collection tube) without wetting the rim. Close the cap, and centrifuge at 20000x g.
Place in a clean 2 mL collection tube.
Carefully open the QIAamp Mini spin column and add 500µL without wetting the rim. Close the cap and centrifuge at 6000x g. Place the QIAamp Mini spin column in a clean 2ml collection tube and discard the collection tube containing the filtrate.
Carefully open the QIAamp Mini spin column and add 500µL without wetting the rim. Close the cap and centrifuge at 20000x g.
Place the QIAamp Mini spin column in a new 2ml collection tube and discard the old collection tube with the filtrate. Centrifuge at 20000x g.
Place the QIAamp Mini spin column in a clean 1.5ml microcentrifuge tube labeled with HBS IDs and discard the collection tube containing the filtrate. Carefully open the QIAamp Mini spin column and add 150µL. Incubate at 56Room temperature for 0h 5m 0s and then centrifuge at 6000x g.
Repeat step 11 for a second elution of 150μl of DNA.
Combine the two 150μl aliquots and mix and split them.
