Mar 16, 2023
Viral PEG purification
- sarah.schulz 1,2
- 1EMBL;
- 2NFDI4Microbiota
- NFDI4Microbiota
Protocol Citation: sarah.schulz 2023. Viral PEG purification. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldp6dxl5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 15, 2023
Last Modified: March 21, 2023
Protocol Integer ID: 78835
Keywords: viral peg purification protocol, viral particle purification, peg percipitation, purification
Abstract
Protocol for viral particle purification via PEG percipitation
Materials
PBS, 0.45 μm syringe filter, 10% w/v PEG-8000, 0.5 M NaCl, 10 mM CaCl2, 50 mM MgCl2, DNase I, RNase A
Troubleshooting
Add 30 ml of ice cold PBS to 3 g of faecal sample and mix by vortexing at high speed for 5 min, then keep on ice
Centrifuge sample at 5000 rpm for 10 min at 4°C and transfer the supernatant to a clean 50 ml centrifuge tube
Filter the supernatant through a 0.45 μm syringe filter
Add 10 % 10% w/v PEG-8000 in saline (0.5 M NaCl) to filtrate, mix by inverting and incubate at 4°C overnight
Centrifuge sample at 5000 rpm for 20 min at 4°C and discard the supernatant.
Re-suspend pellet in 200 μl PBS and transfer it to a 1.5ml Eppendorf tube
Add an equal volume of chloroform to the sample and mix gently before centrifuging it at 2500 rpm for 5 min at room temperature
Transfer the aqueous phase into a new and sterile 1.5 ml tube
Add 40 μl of 10 mM CaCl2 and 50 mM MgCl2 to the sample, as well as 10 U of DNase and 10 U of RNase A
Incubate samples at 37°C for 2 h and then place them in a heatblock at 70°C for 10 min
Store at 4°C