Adenoviral purification by CsCl - Only cell pellet or supernatant too??? (Nov/06/2008 )
How many infected plates do you use if want to purify by CsCl??
A) Do you collect, pellet, discard medium, add small volume of PBS and froze/draw, pellet and collect supernatant, and purify by CsCl? Or….
Infect cell in a small amount of medium, after 90-100% of cytopatic effect, collect cells plus medium, froze/draw, pellet and collect supernatant and purify by CsCl.????
The protocol says to infect 30 T175 flask to have enough for purifying by CsCl doing what I describe above in A, but when I did it (long time ago I did it ones), I don’t remember using so many flask!!! And I’m not sure; I think I did it like B.
Please help!!!!!!
CsCl purification could cause significant viral inactivation. You could try 3% PEG precipitation to purify the virus instead and you would not need to grow 30 T175 flasks. Look for the PEG precipitation in this AquaRNA viral protocol.
Are you sure after PEG precipitation virus are still actives, capable of infect and replicate?????
Because that protocol is for extract viral DNA.
I need virus purified to test in rats. Do you think that this way they are purified enough to don’t cause other reaction in the animals??
How much of infected cell do you recommend me to use?? 2,3, 4 T75 culture flask????
Thanks in advance for your answer
Does anybody know if after PEG precipitation viruses are still actives?? Compared to CsCl purification??
We've always used CsCl purification and virus were perfectly fine afterwards. And yes, we used as much as 40 plates.
Ho Madrius,
Could you provide me your purification protocol please?
Do you discard medium and frozz/draw just the cells suspended in PBS?
40 T75 or T150 or 10 cm plates?? after purification, what is your final volume?
What it the viral title after purificarion?
Thanks in advance for your help!!!