08 November 2013

Genomic-tip, DNA extraction, and protocols

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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

1. Flash freeze ants in 2ml screw-cap tubes, up to 2 Pogo or 3 Aphaenogaster per tube.
2. Add 300ul Buffer ATL to each tube. Crush with pestle.
3. Add one scoop zirconium silicate beads (Quackenbush) to each tube. Blend for 4 minutes at top speed in Bullet Blender (Next Advance).
4. Centrifuge for 2 minutes at 10k rpm.
5. 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.

6. Add 1 ml of Buffer G2 and 50 ul Proteinase K to each tube.
7. Incubate overnight at 50C.

DNA purification

8. Centrifuge tubes 2 min at 10k rpm. Transfer supernatant to a single new 15 ml conical tube leaving behind precipitate.
9. Add 1 ul RNAse A. Incubate for 15 min at 37C.

Following standard Qiagen Genomic-tip protocol.

9. 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).

10. Vortex sample 10 seconds. Pour onto Genomic-tip.

• Take 300 ul aliquot of flow-through (aliquot 2)

11. Wash twice with 5 ml Buffer QC.

• Take 600 ul aliquot of flow-through (aliquot 3)

12. 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!)

13. Precipitate DNA by adding 3.5 ml isopropanol. Mix. Centrifuge for 15 minutes at > 8,000G at 4C (Sorval refrigerated centrigure).
14. Carefully pour off isopropanol supernatant. Add 2 ml cold 70% ethanol. Centrifuge for 10 min at > 8,000G at 4C.
15. Pour of ethanol. Air-dry.
16. Resuspend DNA in 500 ul buffer overnight.