I recruited PEG 8000 (10%) to centrifuge my phage which was incubated for 24h at 4c with PEG, and by 35000g for 50 min. Finally, I had a brown-yellowish thin pellet. It was so low that is was not appropriate for SDS-PAGE or PCR.
I used 5ml of filtered phage and equal PEG. Do I increase the volume of the phage?
Or must I propagate phage (pellet) by incubation in bacteria for 3 or more times?
To precipitate phages, you would add to the supernatant a quarter volume of a solution 20%PEG 8000, 2.5M NaCl; then incubate 1h on ice; and then spin at 10000 rpm for 15 min at 4°C. I don't know what to expect in such conditions you used; also, a colture contamination could lead to absence of phage pellet.
For SDS-PAGE analysis of your phage, you better use a highly purified phage suspension (CsCl-gradient ultracentrifigation is standard practice for this). For a simple PCR, sometimes even crude lysate will do. PEG-precipitated suspensions will do too, of course.
For PEG precipitation, you can look up standard protocols.
Please make sure that your phage amplification was suficiently high. With only a low concentration of phage (
Just to follow up on the previous answer. Once the phage has been precipitated as Paola suggests, a white pellet should be produced Resuspend the pellet in 1-2ml PBS vortex to aid then spin in an ependorf centrifuge at top speed for 5 minutes. The supernatant should be removed. To further clarify repeat PEG precipitation generally a 1/4 or 1/5 dilution with the same method as above. The resulting phage should be much cleaner from bacterial cell debis.
My protocol for M13 phage is:
1. Transfer 320 ml of E coli supernatant to 500 ml bottle and add 80 ml PEG/NaCl (20% polyethylene glycol 8000 - 2.5 M NaCl).
2. Mix well and leave for 1 hour on ice.
3. Spin 3,300xg for 30 min at 4°C.
4. Resuspend each pellet in 15 ml water (or buffer)
From 320 ml E coli supernatant, obtained phage titer is >10^13 cfu/ml in 15 ml.
In our laboratory we use 2 ml of (30%PEG-8000, 3M NaCl) for each 10 ml of clear lysate and preferably keep it overnight at 4oC. Centrifugation is than at 14000 g for 10 min at 4oC.
However the number of phages in the lysate remains crucial for good yield (in our case of phage DNA) so it is important to have phage numbers in the lysate above some threshold (in the case of DNA isolation this would seem to be be >1E09 pfu/ml).
Good luck!
Just go through the link may be u can find the solution
http://www.abdesignlabs.com/technical-resources/bacteriophage-preparation/
Hi,
To concentrate freshwater viruses, I use a protocol established in my laboratory :
http://www.ncbi.nlm.nih.gov/pubmed/17897741
I mix viral concentrated freshwater a solution of 40%PEG/2.4% NaCl in order to have final concentration of 10%PEG/0.6% NaCl in a vial, I let precipitate during minimum 4 days. Then I check for whitish precipitate that floats at the bottom of the vial (Viruses), I take this precipitate very careffully and I put it in tubes to centrifugate them. Then I follow the protocol I attached upper...
1. It is essential to, as pointed out by Zelin Cui, to use a large volume.
2. Add 2-5ml chloroform and shake briefly
3. Centrifuge the lysate to remove debris and unlysed cells
4. Determine the titre - with "good" lytic phages one should expect it to be great than 10>9< pfu/ml
5. Add solid NaCl to 0.6 M, and when dissolved add solid PEG with stirring to 10%
6. Place on ice for 1h or in fridge overnight.
7. Recover precipitate by centrifugation. Please note that with Pseudomonas aeruginosa phages you can expect a very large pellet.
8. This must be cleaned-up further, ideally by CsCl equilibrium centrifugation prior to SDS-PAGE.
We have recently stopped using PEG in a lot of cases and just pellet the phage directly in a centrifuge with a high-capacity rotor (Sorvall GS-3 or GSA, Beckman JA-10, etc.). Pelleting time, T (in hours), is equal to K/S, where K is the rotor K-factor and S is the sedimentation coefficient (in Svedbergs). K factors are usually listed in the centrifuge rotor manuals, and you can estimate S based on your phage: T7 is about 500 S, lambda is about 600 S and T4 is about 800 S. I'm not sure what the S value is for M13. In general I like to spin the phage about 2-3x the pelleting time to make sure I compensate for viscosity and temperature.
So, if you have a Sorvall GSA rotor, you could pellet up to 1.2 L of a phage like T7 or lambda by centrifuging it at 6,000 x g, 4 deg. C for 12 - 18 hrs (or 10,000 x g, 4 deg. C for 8-10 hrs). Dump the supernatant, add new buffer and let the pellet soak in the fridge for at least a few hours before you try to resuspend it, if you are too aggressive with the resuspension the phage might fall apart.
I've found this gives a much cleaner final product than PEG and it is often faster as well, as long as nobody minds you monopolizing the centrifuge for a whole day.
Take a look of Yamatomo et al. Virology 40: 734-744 (1970) This is an old paper, but it is the basic stuff.
Dear Reza,
I just saw your question only now (too busy with writting !!! Please read my article entitled : Armon, R., Arella, M. and Payment, P. A highly efficient second-step concentration technique for bacteriophages and enteric viruses using ammonium sulfate and Tween 80. Can. J. Microbiol. 34: 651 – 655, (1988). I garantee that it works much better than all other methods !!
Robert
Check the titer of amplified phages, if it's 10^12-10^14 pfu/mL, it's working fine. Just scale up the prep and you would get enough for running on SDS-PAGE. For PCR though, I think the current volume of phage should be sufficient, provided the titer is right. Overnight precipitation with 4% PEG-8000+ 400 mM NaCl (final concentrations) at 4 degC works fine. Generally, infection at OD600 of 0.5 and amplification with vigorous shaking for 4-5 hours at 37 degC is good for M13 phage amplification.
by adding NaCl phage will separate(membrane attach phage) from bacterial membrane....
Nabanita Giri.
I have a question regarding this matter.I am trying to precipitate the 10^14 pfu/ml (70ml) phage,the PEG precipitation protocol is same as you have mentioned but the pellet which is precipitated after centrifugation is so hard that it is not dissolved in 0.1 M kcl buffer, and so ultimately I am not getting the concentrated phage solution. Please help me.
@Utpal Basu,
Try centrifuging less long or with reduced speed...
See if that will give you a weaker pellet.
Dear Expertise,
I followed all your advice, I didn’t get precipitation during the mix (invert), only I got pellet after the centrifugation and I have doubts whether this pellets are precipitated phage’s or PEG/NaCl.
-stalin
Dear all,
After concentrating phages using PEG precipitation, how to remove the PEG from the phage particles? Do we have to dialyse or is there some other method?
To remove PEG, add the same volume of chloroform. Invert. Centrifuge in a benchtop centrifuge at max speed for 5 min.
Normally, the bottom layer is chloroform, the top layer is your phage suspension. Inbetween there is a layer of PEG. When you remove the top layer, you have removed a lot of the PEG.
Make sure the phages you are using are not sensitive to chloroform!
Hiii. I have phage 5' for E.coli ATCC 13706. I tried to increase phage concentration by method: 50 ml phage 108 PFU/ml + 10 ml of E.coli + 150 ml of PYCa medium, after incubating at 37 oC for 12 h, little clear medium can be seen, and the concentration of phage is around 108 PFU/ml.
Do you know any method to increase phage concentration to 1011 or 1012 PFU/ml? please let me know. Thank you very much.
You have to inoculate phage at an M.O.I : 0.001 to 0.00001 and use the log phase E.coli culture. With this method you may increase the titre up to 1012 to 1014 pfu/ml. MOI is crucial. Best wishes.
Hi Utpal,
DO you know how long I have to incubate to get 10^12 PFU/ml (I will use 100 ml log phase E.coli OD 1 + 1mL phage 10^6 pfu/ml)? And do you know any symbol to recognize 10^12 pfu/ml? Thanks
Hi Son,
In a perfect situation, the growth medium of your E. coli culture will become more clear again, indicating that a lot of the bacterial cells have died. This normally happens about three to six hours after you have added the phage. At this point you can harvest the newly grown phage. If you let the bacterial culture incubate longer, it will become blurry again, but no extra phage will have grown.
An OD (at 600nm) of 1 is really high, though. Grow the cells until OD600 = 0.6. Then infect with the phage. It all depends a bit on the phage and the bacterium. Play around with OD and MOI until you find something that works.
Hi everybody, I have two Aeronmonas salmonicida phages, which grow very poor in broth (I can only get 104pfu/ml). So I used agar plates to propagate them, which can give me 107 puf/ml. I want to sequence their genome, however, with this low concentration, I cannot get high enough concentration of DNA. Could anybody tell me, in which way I can concentrate the phages, or concentrate the genome DNA for at least 100 times, so that the samples can be qualified for sequencing?
Many thanks.
Hi Reza
Its better to increase the incubation time of the phage-containing bacterial host for one extra overnight. sometime we incubate the bacteria for 72 h incubation.
Increasing the incubation time lead to more phage production.
And others said its better that the final concentration of PEG be 20%. And also the ratio of PEG8000 solution to phage must be about 1:4.
Follow this Standarized protocol :Use 10 % PEG and overnight incubation and then centrifuge at 14000 RPM for 2 hrs.breakdown the pellet ,that would contain phages
For best precepitation, I use PEG 8000 4% / NaCl 3% (250ml culture) and incubate it for 1 hour at 4°C. this will give you the maximum yield of phages.
the yellowish pellet may contains some bacteria, but you can eliminate them after resuspension of phages with maximum speed centrifugation (14000g, 10 minutes).
keep in mind that resuspend the phages carefully, as the phages can be broken during resuspension.
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