1M NaCl doesn't dissolve...not sure why - PEG precipitation of phages (Sep/30/2008 )

Hi there,

I just started working with phages and wanted to concentrate them by PEG precipitation. And, of course, trying this protocol for the first time, something doesn't work quite right as it possibly should.

I collected water at a spring (fresh-water), used Tangential Flow Filtration (30kDa membrane) to concentrate the sample, separated the viruses from the bacteria with a 0.2um filter and now I need to concentrate the viral solution more. So I decided to do PEG precipitation. Unfortunately, I got kinda stuck at the step where NaCl is added, because the salt doesn't dissolve (it looks like a milky solution).

here is what I did so far:
1. added 10ml chloroform to 200ml of viral spring solution...final conc. 0.5% (to kill any bacteria that are still in the solution)
2. incubated for 30min, RT and 100rpm
3. added DNase and RNase
4. incubate it overnight in the dark, RT
5. added 11,7g of solid NaCl to bring the solution to 1M
....but it doesn't dissolve....the solution looks milky
....I moved the solution into 37C, shaking at 100rpm (for over an hour now), but it still looks the same

I did a separate test with the spring water from the same source (without organisms...filtrate from the TFF filtration): 50ml of water and 2.92g of NaCl....dissolved right away. That means to me that the water of "environmental sample" is not the reason for the "not-dissolving" of the salt.

I am at a point where I can't explain what happened and I am running out of ideas. I would be grateful for any input on what to do or what I possibly did wrong. Thanks!

Cheers,
Verena

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Are you sure it isn't dissolved - are you getting a precipitate of crystals at the bottom of the solution?

DNA and protein will precipitate out of solution at high concentrations of salt. It could be this that you are seeing, or some sort of chloroform/salt/protein mix that is cloudy.

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It eventually dissolved after 2.5 hours at 37C, shaking.....then the solution was clear. I was kinda relieved that it did, but now the next problem came up. I added PEG8000 (10%) last night, put in the fridge, but I can't see any kind of precipitation or "cloudiness".....Now where it needs to be milky looking, it's not.

Thanks for your help,
V

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