Phage gDNA extraction (Qiagen DNEasy kit)
Materials required
- Phage Lysate: 500uL high titer (~\(10^{10}\)), filtered with 0.22uM filters
- DNAse I: 1U total
- RNAse A: 1uL of 10mg/mL (0.01 mg total)
- DNAse buffer: 50uL of 10X
- EDTA: 20uL of 0.5M
- Proteinase K: 1.25 uL of 20mg/mL
- DNAeasy Blood & Tissue Kit (Buffers AL, AW1, AW2, AE)
- DNA Wash Buffer: 80% EtOH + Tris-HCL
- 100% EtOH
- Nuclease free H2O
Procedure
- Add 50uL of 10X DNase I Buffer to 450uL lysate in a 2mL DNA Lobind tube.
- Add 0.5uL of 2U/uL DNASE I and 0.5uL of 20mg/mL RNAse A and incubate at 37C for 1.5h (without shaking).
- Add 20uL of 0.5M ultrapure EDTA to inactivate DNAse I and RNAse A (mix gently)
- Add 1.25uL of 20mg/mL Proteinase K to the mix, mix gently, and incubate at 56C for 1.5h (without shaking).
- Add 523uL AL Buffer to the treated ~523 ul treated phage lysate, vortex, and incubate at 70C for 10 minutes.
- Add 523 uL 100% EtOH and vortex to mix.
- Transfer 750uL of the mix to a DNeasy Mini spin column in a 2mL collection tube and centrifuge for 1 minute at 6000x rcf. Discard flow through. Do it multiple times until all the liquid has been passed through the column, discard flow-through and collection tube.
- Place the column in a new 2mL collection tube, add 500uL buffer AW1 making sure to wash the walls of the tube in the process, and centrifuge for 1min at 6000x rcf. Discard flow through with collection tube.
- Place the column in a new 2mL collection tube, add 500uL buffer AW2 making sure to wash the walls of the tube in the process, incubate at RT for 5 minutes, and centrifuge for 3min at 20,000x rcf to dry the membrane. Discard flow through and collection tube.
- Place the column in a new 2mL collection tube, add 750uL DNA Wash Buffer making sure to wash the walls of the tube. Centrifuge at 20,000x rcf for 3 min to dry the membrane. Discard flow-through.
- Centrifuge again for 1min at 20,000x rcf to make sure there's no carry over buffer left.
- Place the DNeasy spin column in a sterile 1.5mL DNA LoBind tube and elute with 50uL AE Buffer or NF water (heated to 65C, incubate the tube with the water/buffer at 65C for increased yield for 5 minutes).
- Spin the column at 6000x rcf for 1 minute to elute DNA
- Do a second elution with 30uL of buffer/water to increase yield even further. Store at 4C.