Manual Gentra Puragene DNA Extraction
Clemens Scherzer, Bradley Hyman, Charles Jennings
Abstract
This protocol explains the Standard Operating Protocol for manually extracting DNA using Gentra Puragene.
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 Gentra Puregene DNA Extraction
Preheat water bath to 37°C before removing whole blood samples from -80°C freezer.
Thaw whole blood samples by gently agitating sample in the 37°C water bath. Once thawed, reset water bath to 65°C for last step in protocol.
Add 9mL to a 15 mL centrifuge tube.
Add 3mL and mix by inverting 10 times.
Incubate sample for 0h 5m 0s at 65Room temperature (15°C-25°C). Invert once during the incubation.
Centrifuge at 2000x g,25°C.
Pour off and discard supernatant. Leave approximately 200 µL of residual liquid and the white blood cell pellet.
Vortex tube on high speed to resuspend pellet in the residual liquid.
Add 3mL. Vortex on high speed for 0h 0m 10s.
Add 1mL. Vortex on high speed for 0h 0m 20s.
Centrifuge sample at 2000x g,25°C.
Add 3mL into a clean 15 mL tube. Carefully pour the supernatant from step 11 into this tube.
Invert samples 50 times until DNA is visible as threads or a clump.
Centrifuge tube at 2000x g. Observe DNA as a small white pellet.
Carefully pour/pipette off supernatant without disturbing the pellet. Drain excess drops of supernatant by inverting on a Kimwipe, while making sure that pellet remains in the tube.
Add 3mL and invert 3-5 times to wash the DNA pellet.
Centrifuge pellet at 2000x g,25°C.
Carefully pour/pipette off ethanol without disturbing the pellet. Drain excess drops of ethanol by inverting on a Kimwipe, while making sure that pellet remains in the tube.
Air dry the pellet for 0h 1m 0s.
Add 250µL. Vortex on medium speed for 0h 0m 5s.
Incubate DNA in 65°C water bath for 1h 0m 0s in order to dissolve DNA.
Incubate at 65Room temperature (25°C) 1h 0m 0s on rocker.
On the following day, DNA can be transferred to a 1.5 mL tube. Split volume in half to create two aliquots of DNA (DNA0-01 and 02).
Aliquot 3µL into a 1.5 mL tube for Cary concentration assay. (Store in -20°C if not being assay immediately.)
Aliquot 1.5µL into a PCR tube for .7% agarose gel electrophoresis to confirm presence and size of DNA. (Store in -20°C if not being assay immediately.)
