I'm currently doing phage display technology using the NEB phage display kit. As instructed by the manual, i have to precipitate the phage in supernatant by centrifugation (12 000xg, 4C). However i didn't have access to such a high-speed centrifuge. The only one we got here is 5000 rpm centrifuge. I didnt' get any pellet/white residue when centrifuging the supernatant at 5000 rpm. May i know if you have any alternative to PEG precipitation or simply any alternative to high-speed centrifugation to precipitate my phage?
FYI, the phage supernatant was incubated with 1/4 volume of 20% PEG-8000/2.5M NaCI overnight at 4C.
you can use Amicon Ultra centrifugal filters instead of PEG Nacl method. Amicon 4K will help. these filters operate at 5000rpm for about 5 to 10 minutes.
Iqbal Alvi Can I use Amikon ultra centrifugal filters for concentrating viral particles from the samples that has low viral load.
Hi Fatimah, the condtions you used should normally work well even with a centrifugation step at 5000rpm. if you have no pellet in these conditions you should wonder if there was production of phages, meaning are you sure that the infection step was OK. If not, no phages will be produced and you will have no pellet. Even if the pellet is not big, you should have a close look at your centrifugation tubes, as the pellet might be smeared out over the lenght of the tube. As suggested by other collegues you can also use ultracentifugal filters.
Hey Fatimah.
Even I was working on the same aspect and was trying to concentrate viral particles from the sample which already has lower microbial load.
During this, I came across a method which uses Zinc chloride to concentrate viral particles. My aim was to extract NA specifically from VIRUS/FREE PHAGE
The trick is if you do not want to extract nucleic acid then you would have to stop after first step (after adding ZN CHLORIDE)and try to purify it.
But if you want to extract NA then follow the entire protocol, however, try to increase ethanol washes.
It is a very short method and less time consuming especially for extracting NA from viruses (Provided that you filter with 0.25 micron, if it is not a pure lysate)
sending you the link, hope it helps.
https://academic.oup.com/nar/article-abstract/19/19/5442/1027023?redirectedFrom=PDF
Dear Rakesh Kumar yes you can but it will increase the titer by almost 2-3 folds.
I am curious about the results from Amicon Ultra filters. Did anyone use them for M13 phage purification? If so, can you share a brief procedure ?
Thanks!
Dear Kiran S Gajula i have used Amicon Ultra filters 4k for phage purification of tailed phages.i think for M13 phages 3K will work.
Dear Iqbal Alvi ...Thanks for your response. I am relatively new to practice PD. So the idea is to just centrifuge the supernatant after infection and then use the concentrated phage for downstream steps such as phage titration, panning and amplification? Does this mean that I don't have to use PEG and NaCl? Please advice..Thanks