Qiagen Genomic-tip extraction for ants
Protocol to extract high-molecular weight DNA using Qiagen Genomic-tip Midi Kit. Note that using the “Tissue” prep with only Buffer G2 for homogenization and lysis did not work and a cell lysis buffer (I use the Buffer ATL supplied with the DNeasy kits) is necessary.
Tested for Pogonomyrmex and Aphaenogaster ants.
Protocol below is for 20-30 ants in 10 screw cap tubes. Modify for more or fewer.
Tissue homogenization and cell lysis
- Flash freeze ants in 2ml screw-cap tubes, up to 2 Pogo or 3 Aphaenogaster per tube.
- Add 300ul Buffer ATL to each tube. Crush with pestle.
- Add one scoop zirconium silicate beads (Quackenbush) to each tube. Blend for 4 minutes at top speed in Bullet Blender (Next Advance).
- Centrifuge for 2 minutes at 10k rpm.
- Transfer supernatant from each tube to a new 1.5 ml centrifuge tube. Avoid transferring debris by using a P200 then P20 (small tip prevents debris transfer).
- Alternatively, if centrifuge for 15 ml conical tubes and water bath available, combine all supernatant in single 15 ml conical. Follow next two steps with 10x volume in single tube.
- Add 1 ml of Buffer G2 and 50 ul Proteinase K to each tube.
- Incubate overnight at 50C.
- Centrifuge tubes 2 min at 10k rpm. Transfer supernatant to a single new 15 ml conical tube leaving behind precipitate.
- Add 1 ul RNAse A. Incubate for 15 min at 37C.
Following standard Qiagen Genomic-tip protocol.
- Equilibrate Genomic-tip with 4 ml Buffer QBT. Allow all buffer to flow through.
- Take 300ul aliquot of sample and save for analytical gel (aliquot 1).
- Vortex sample 10 seconds. Pour onto Genomic-tip.
- Take 300 ul aliquot of flow-through (aliquot 2)
- Wash twice with 5 ml Buffer QC.
- Take 600 ul aliquot of flow-through (aliquot 3)
- Place Genomic-tip over 15 ml glass tube for Sorval centrifuge. Elute DNA with 5 ml Buffer QF.
- Take 300 ul aliqot of flow-through (aliquot 4 - should contain DNA!)
- Precipitate DNA by adding 3.5 ml isopropanol. Mix. Centrifuge for 15 minutes at > 8,000G at 4C (Sorval refrigerated centrigure).
- Carefully pour off isopropanol supernatant. Add 2 ml cold 70% ethanol. Centrifuge for 10 min at > 8,000G at 4C.
- Pour of ethanol. Air-dry.
- Resuspend DNA in 500 ul buffer overnight.