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)
- 100% EtOH
- Nuclease free H2O
Procedure
- Add 50uL of 10X DNase I Buffer to 450uL lysate in a 2mL DNA Lobind tube.
- Add 1uL of 1U/uL DNASE I and 1uL of 10mg/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 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 and centrifuge for 3min at 20,000x rcf to dry the membrane. Discard flow through and collection tube. Centrifuge again with a new collection tube 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 30uL AE Buffer or 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.