PEG didn't precipitate right away....what is your experience or advice? - PEG precipitation (Oct/02/2008 )

Hi there,

I am trying to concentrate phages from an environmental freshwater sample by PEG precipitation. Unfortunately it doesn't work as I would have hoped to....the PEG doesn't precipitate. I am using the protocol from Sambrook, but I am not sure what I am doing wrong.

The short protocol is:
1. kill bacteria with chloroform (0.5% final concentration), incubate at 37C
2. add DNase and RNase, incubate at RT
3. add NaCl to 1M final conc., incubate on ice
4. centrifuge
5. add PEG8000 to final conc. of 10%, incubate at 4C

....and that's where I get stuck. The PEG doesn't precipitate. The protocol suggests a incubation time of 1hour, but my samples are in the fridge already for over one day and nothing seems to happen. I also tried a sample without adding chloroform in the first place or another completely different sample. ....HELP.....

Do you guys have any suggestions on what I could do or what I am doing wrong? I would be grateful for any advice or input. Thanks!

Cheers,
Verena

Friday, 3OCT08

Hi there,

so, the PEG finally seems to precipitate. I would like to hear your experience and opinion about PEG precipitation, especially because I am just starting to learn this methods and I am not sure why it took so long. I have some questions:

-What are your experiences with PEG precipitation?
-Does the solution has to look very milky or cloudy.....or is a little bit normal?....it seems like that the PEG in my sample seems to precipitate, but it doesn't look very milky more like light white faint "streams" when I shake it (if you know what I mean)
- How does particle concentration related to PEG concentration? If you have less particles should you use less PEG?
- What is a normal time span for the precipitation....is it normal that it takes 2 days instead of 1 hour as the protocol suggests?
- Are there any steps in the protocol that you think are very critical and deserve attention?....I am worried that maybe didn't pay enough attention to certain steps that might be important

Please let me know what you think!

Cheers,
Verena

---

Preparing PEG solution needs strong vortex and warm. It will not easily dissolve in room temperature...in your case it is 4oC some more. But i am not sure if chloroform or NaCl will accelerate it...unlikely.

I have never gone through the protocol, but i guess it means PEG solution instead of PEG powder? Please confirm it.

I used to prepare 90% PEG solution by vortexing, and alternate warming it in 37oC waterbath. It dissolve nicely.

---

Did you centrifuge your sample after adding PEG8000 and refrigerating it?

If no, please do so.

If yes, it means the DNA in your sample is too dilute for the PEG to precipitate it. Increase the amount of bacteria that you are working on.

---

No, I didn't centrifuge it after I dissolved the PEG.....why is it important? Please let me know.

Thanks for your reply!

---

The PEG dissolved well in the solution, but it didn't precipitate. I finally can see something after 2 days, but I am still a little bit worried because it took so long.

Thanks for your reply!

---

Hi there,

so, the PEG finally seems to precipitate. I would like to hear your experience and opinion about PEG precipitation, especially because I am just starting to learn this methods and I am not sure why it took so long. I have some questions:

-What are your experiences with PEG precipitation?
-Does the solution has to look very milky or cloudy.....or is a little bit normal?....it seems like that the PEG in my sample seems to precipitate, but it doesn't look very milky more like light white faint "streams" when I shake it (if you know what I mean)
- How does particle concentration related to PEG concentration? If you have less particles should you use less PEG?
- What is a normal time span for the precipitation....is it normal that it takes 2 days instead of 1 hour as the protocol suggests?
- Are there any steps in the protocol that you think are very critical and deserve attention?....I am worried that maybe didn't pay enough attention to certain steps that might be important

Please let me know what you think!

Cheers,
Verena

---

Umm... how do you intent to pellet the viral DNA once it has precipitated in PEG? All you get will be a suspension.

---

yes, I would pellet the phages by centrifugation. I read in several protocols that one has to wait with the centrifugation till one sees a milky appearance in the solution, which is an indicator that the phages together with the PEG precipitate... but that didn't happen for two days....I didn't see any white particles....the solution just stayed clear. I was worried because nothing happened for a long time. I apologize for the confusion.....I thought you meant that I had to centrifuge right away after I added the PEG (before any "white stuff" was visible).

---

Verena, PEG would not precipitate by itself and if the phage (and other particles) concentration was low in your water sample, you might not see the precipitation. Just go ahead spin it and it will come down.

Here is a simple and quick protocol for concentrating phages (and any viruses) and extracting viral DNA, RNA, and proteins.

AquaRNA Viral Protocol

AquaRNA is suitable for extracting DNA, RNA, and proteins from double- or single-stranded DNA viruses and RNA viruses, including bacteriophages. The DNA, RNA, and protein yields may vary depending on the genome size of the virus and the viral titer of the starting material but are close to theoretical values. The starting volume of 50 ml used in this protocol is for demonstration only; you may use different starting volume. For example, you may process 1 ml of bacterial culture containing 10^9 phage virions with 50 ul of AquaRNA to recover ~2 ng DNA from phi X174 or ~120 ng DNA from T4, sufficient for most PCR analyses.

1. Centrifuge virus infected culture (50 ml) at 12,000g for 5 min to pellet the cells.

2. Transfer the virus containing supernatant to a new centrifuge tube.

3. Add 1 volume of 20% PEG8000 in 2.5M NaCl to 5 volumes of virus supernatant.

4. Vortex to mix and centrifuge at 12,000g for 10 min to pellet the virions.

5. Decant or aspirate to remove the PEG supernatant as complete as possible.

6. If the virus is to be saved as high titer stock, suspend the pellet in 100 ul of 10% glycerol in PBS and store at -20 °C. Otherwise, add 0.5 ml of AquaRNA solution to the pellet and proceed to next step to extract the DNA (or RNA).

7. Vortex vigorously and incubate on ice for 15 min to lyse the virions.

8. Transfer the lysate to a 1.5-ml microfuge tube and add 1 volume of isopropanol.

9. Vortex to mix well and centrifuge at 12,000g for 5 min to pellet the DNA (or RNA).

10. If viral proteins are to be recovered, transfer the protein containing supernatant to a new tube for protein extraction later. Otherwise, decant to discard the supernatant.

11. Rinse the viral DNA (or RNA) pellet with 75% ethanol 3 times by filling the tube with the ethanol solution from a squirt bottle and then decanting to discard the solution.

12. Air-dry the pellet and dissolve the DNA (or RNA) in 50 ul of water or TE buffer.

---