Oropharynx gDNA Extraction in Microcentrifuge Tubes

Buffer Preparation

• Lysis Buffer: Ready to use.
• Binding Buffer: Add 17 mL isopropyl alcohol to the DNA Binding Buffer bottle.
• Wash Buffer 1: Add 22.5 mL of 100% ethanol to the Wash Buffer 1 bottle.
• Wash Buffer 2 (75% ethanol): 3 parts of 100% ethanol to 1-part of molecular grade water, prepare fresh each day, producing 1.4 mL per sample with 10% excess.
• DNA Elution Buffer: Ready to use.

Enzyme Reconstitution

Proteinase K:

• Bring proteinase K vial to room temperature.
• Remove stopper and add 1.1 mL of molecular grade water to the proteinase K vial.
• Replace stopper and incubate at room temperature for 15 minutes, mixing well by inversion.
• Aliquot into required volumes and store at -20°C. Avoid multiple freeze thaw cycles

RNase A:

• Bring RNase vial to room temperature.
• Remove stopper and add 500 μL of molecular grade water to the RNase vial.
• Replace stopper and incubate at room temperature for 15 minutes, mixing well by inversion.
• Aliquot into required volumes and store at -20°C. Avoid multiple freeze thaw cycles.

Homogenization

1. Weigh up to 50 mg of tissue samples and transfer to lysis tubes.
2. Set the Bullet Blender at speed 12, time 3 minutes and homogenize the samples dry. If using other homogenizer models, refer to the manufacturer's instructions for appropriate settings.
3. After Homogenization, add 500 μL of Lysis Buffer, 20 μL of proteinase K, and 10 μL of FoamBlocker into each lysis kit tube.
4. Set the Bullet Blender at speed 12, time 1 minute and homogenize the samples wet.
5. Remove the tubes and visually inspect the samples to confirm complete homogenization (Figure 1).

Extraction

RNase Treatment & Binding:

1. Prepare fresh microtubes in the tube rack with 200 μL of PBS.
2. Transfer 320 µL of the homogenate to the new tubes containing PBS.
3. Add 5 µL of RNase A to each sample and slide the tube rack onto the mixing base. Mix well by inversion and leave at room temperature for 10 minutes, mixing again halfway through the incubation.
4. Centrifuge tubes at 13,000 x g for 5 minutes.
5. Transfer 450 μL of supernatant into new microfuge tubes, being careful not to disturb the pellets or transfer any debris.
6. Add 450 μL (or volume equal to the transferred supernatant from step 4) of Binding Buffer to the supernatant in each tube. Slide the tube rack onto the mixing base and inverse to mix the buffer with the homogenate.
7. Vortex the MAGneat beads thoroughly making sure that there are no clumps in the tube.
8. Slide the tube rack off the mixing base and add 30 μL of the beads to each sample tube. Mix tubes by inversion to resuspend the magnetic beads. Allow to stand for 5 minutes, mixing again halfway through.
9. Slide the tube rack onto the magnetic stand and allow to sit for 1-2 minutes, until the magnetic beads separate from the homogenate (Figure 2).
10. Using a pipette, discard the supernatant without disturbing the beads (Figure 3). Remove the tube rack from the magnetic stand.

Washing:

1. Add 700 µL of Wash Buffer 1 to the tubes and then place the tube rack in the mixing base. Mix thoroughly by inversion, ensuring all beads have been detached from the tube walls and evenly suspended (Figure 4).
2. Remove the tube rack from the mixing base and place it on the magnetic stand. Allow the magnetic beads to separate.
3. Using a pipette, discard the supernatant.
4. Remove the tube rack from the magnetic stand. Add 700 µL of Wash Buffer 2, mix thoroughly by inversion to completely resuspend the beads.
5. Transfer the tube rack to the magnetic stand and allow the magnetic beads to separate.
6. Discard the supernatant.
7. Repeat steps 4-6 for a total of 2 washes with Wash Buffer 2.
8. Centrifuge samples at 2,000 x g for 3 minutes to collect the remaining Wash Buffer. Place the tubes on the tube rack.
9. Use a 20 µL pipette to remove the remaining Wash Buffer from the tubes (Figure 5).
10. Leave tube caps open to air dry the magnetic beads at room temperature for 5 minutes.

Elution:

1. Add 100 µL of DNA Elution Buffer to each tube. Mix by tapping to ensure that the magnetic beads are completely resuspended.
2. Incubate at 60°C for 5 minutes with caps closed.
3. After incubation, place the tube rack back onto the magnetic stand to allow the beads to separate.
4. Transfer the clear supernatant containing the gDNA into new, labeled microfuge tubes for downstream processing. Be careful not to transfer any magnetic beads.
5. Analyze quality (OD₂₆₀/OD₂₈₀) and yield using a NanoDrop™ or other spectrophotometer and agarose gel.
6. Isolated DNA can be stored at 4°C for up to a week or at -20°C for long term storage.