VLP Extraction from Fecal Samples
Frej Larsen
Abstract
This protocol is for isolation of bacteriophages from fecal matter. It is based on the protocol used for the COPSAC10 cohort and was originally described by Ling Deng in doi:10.3390/v11070667. Extraction is performed using centrifugation and ultrafiltration to isolate viral particles and get rid of contaminants.
Before start
Fill bucket with crushed ice
Turn on centrifuge and start the fast temp program to reach 4◦ C
Cool SM buffer in ice bucket
Steps
Thaw frozen sample in ice bucket
Transfer up to 500mg of sample to centrifuge tube and add SM buffer to reach a final volume of 5mL. Place centrifuge tube back in ice box
Homogenize samples by placing ice box on shaking board at 300rpm
Centrifuge samples at5000rcf,4°C
Filter the supernatant through a 0.45 um PES filter into a new centrifuge tube
Transfer 2.5mL of the sample to the outer chamber of the concentrator
Centrifuge sample in the concentrator at 1500rcf,4°C
Pour off liquid from the inner chamber
Repeat the three previous steps until all liquid from the centrifuge tube has passed through the concentrator and the there is no liquid left in the inner chamber
Add 100 ul SM buffer to the inner chamber and centrifuge 1500rcf,4°C
Leave in fridge to release VLPs from filter for at least 12 hours and at most for 1 week before proceeding with spotting or DNA/RNA extraction
