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2026-04-29 18:03:32 Bhaskar Kumawat: Preliminary protocol
phage gdna extraction (pci).md ..
@@ 1,23 1,27 @@
- # Phage gDNA extraction (PCI)
+ # Phage gDNA extraction (Qiagen DNEasy kit)
## Materials required:
-
- ### For phage lysis
- - Phage lysate: 500uL high titer, filtered with 0.22uM filters
- - DNAse I: 10U total
- - RNAse A: 1uL of 100mg/mL (0.1 mg total)
+ - 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: 2.5U total
- - SDS: 25uL of 10%
-
- ### For DNA extraction
- - TE-saturated Phenol:Choloroform:Isoamyl alcohol 25:24:1
- - Chloroform:Isoamyl alcohol 24:1
+ - Proteinase K: 1.25 uL of 20mg/mL
+ - DNAeasy Blood & Tissue Kit (Buffers AL, AW1, AW2, AE)
+ - 100% EtOH
+ - Nuclease free H2O
+ ## Procedure
+ 1. Add 50uL of 10X DNase I Buffer to 450uL lysate in a 2mL DNA Lobind tube.
+ 2. Add 1uL of 1U/uL DNASE I and 1uL of 10mg/mL RNAse A and incubate at 37C for 1.5h (without shaking).
+ 3. Add 20uL of 0.5M ultrapure EDTA to inactivate DNAse I and RNAse A (mix gently)
+ 4. Add 1.25uL of 20mg/mL Proteinase K to the mix, mix gently, and incubate at 56C for 1.5h (without shaking).
+ 5. Add 525uL AL Buffer to the treated ~522 ul treated phage lysate, vortex, and incubate at 70C for 10 minutes.
+ 6. Add 525 uL 100% EtOH and vortex to mix.
+ 7. Transfer 750uL of the mix to a DNeasy Mini spin column in a 2mL tube (do it multiple times until all the liquid is through)
+ 8.
## Sources
- [https://www.pnas.org/doi/10.1073/pnas.2104592118#sec-3](https://www.pnas.org/doi/10.1073/pnas.2104592118#sec-3)
- [At the Bench: A Laboratory Navigator (pg 284, pg 145)](https://www.cshlpress.com/default.tpl?cart=1777472107764588063&fromlink=T&linkaction=full&linksortby=oop_title&--eqSKUdatarq=470)
+ [https://www.mdpi.com/2409-9279/1/3/27](https://www.mdpi.com/2409-9279/1/3/27)
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