Dear Pallavali

Do you have any problem if using phage DNA extraction kit ?

the link below may help your research requirement 

or there is another method but you should perform it manually and may be time-consuming by using chloroform , phenol , SDS and other materials , in this way you can obtain > 500 ug DNA as this link explain full detailed steps

i'll attach it for you 

https://norgenbiotek.com/product/phage-dna-isolation-kit

http://vlab.amrita.edu/?sub=3&brch=186&sim=1377&cnt=2

Dear Palavalli: It depends on your bacteriophages. Here are the methods you may try:

Characterization of a virulent bacteriophage LK1 specific for Citrobacter freundii isolated from sewage water.   Waqas Nasir Chaudhry · Irshad Ul Haq · Saadia Andleeb · Ishtiaq Qadri ·Journal of Basic Microbiology 06/2014; 54(6). DOI:10.1002/jobm.201200710 · 1.82 Impact Factor

Isolation and Partial Characterization of a Virulent Bacteriophage IHQ1 Specific for Aeromonas punctata from Stream Water Irshad Ul Haq · Waqas Nasir Chaudhry · Saadia Andleeb · Ishtiaq Qadri ·Microbial Ecology 09/2011; 63(4):954-63. DOI:10.1007/s00248-011-9944-2 · 3.12 Impact Factor

Hi Pallavali,

As somebody already told you above, it depends on the bacteriophage family but in summary, you need to pellet the phages in high concentration, 3*10^4 PFU (30000) is a little low. In lactic acid bacterias (LAB) we first make a precipitation with PEG (or ultracentifugation on CsCl gradient) and then start with a kit like Lambda kit of Qiagen or the old protocols with phenol/chloroform...

I attach you the protocol we use with the Lambda kit modifications for LAB...

Good luck!!

Alfonso

• kit was costly, so  need manual method for isolation of DNA from phages.
• i followed the concentration of  phages by PEG.
• treated with proteinase K and with nucleases
• next followed the chcl3 and phenol method 
• it given good results
• thank you all ..................

Tq katarzyna, its about three years back question..I isolated DNA and now I am in thesis writing part dear..Tq for ur suggestion..I was isolated Bacteriophage belonging to three families I.e, siphoviridar, myo and podoviridae..theses phages were used as antibacterial agents against septic wound causing bacterial biofilm..

Tq dear ...

Dear Katarzyna Waniolka,

Thank you dear for ur congrats. yes host range is very import factor to use phages as bio-control agents.I isolated 4 different bacteriophages belonging to Siphoviridae, Podoviridae and Myoviridae family members for the eradication of biofilms of MDR- S. aureus, P. aeruginosa, E. coli and K. pneumoniae separetely..Phage genome Sequencing is cost effective one..so..i didnot done that one dear...

Dear all I also currently work with phage DNA extraction by using promega clean up but I keep getting very low DNA concentration 18 -90 ng/ul .even after trying different modification .pallavali the manual DNA extraction in first comment work with you?

and if you did RFLP for phage can you send the protocol .

thank you

any way thank you Katarzyna Waniolka , I read promega clean up is not suitable for all phages

Try only method below given ,you will get great help

1Centrifuge mixture at 4000 RPM

2.Filter supernatant with 0.45 u filter

3. Centrifuge filtered content at 10 000 RPM and transfer supernatant to another tube.

4. Add 10 % PEG 8000 and incubate at 4 c for 12 hrs.

5. Do centrifuge at 50000 rpm for 1.5 hr

6.Discard Suprnatant and add sm buffer to the pellet .

7. Again do ultacentifuge at 50000 rpm for 1.5 hr

8.repeat the step no. 6 a

9. Now prefer Norgen (canada) to isolate DNA

You can use this mixture for following protocols

a)TEM

b)DNA sequencing

c)In vivo experiments

Dear Pallavali,

Pls share your protocol

Thanks

@pallavali hope you got your answer

Naveen Chaudhary

During PEG treatment of bacteriophages from bacterial lysate(Filtered), do we also get Bacterial DNA contamination. as PEG is also used to precipitate DNA

Hi Nisha,

It will be helpful for your research, please go through once... 

Extraction of genomic DNA from bacteriophages

 20ml of high tittered Vibrio phage containing SM buffer suspension (10 7–10 10 PFU/ml) treated with 10 µl RNase A (5 mg/ml) and 10 µl DNase I (10 mg/ml) were incubated 37˚C overnight.  After the incubation 8 ml precipitant solutions (33% PEG800, 3.3 M NaCl) was added and gently mix well, the mixture was stored at -80 for 1 hour. Then centrifuged at 14000 rpm for 45 min, 4˚C the supernatant was removed and precipitate pellet resuspended in 5ml of SM buffer and gently mixed by pipeting and transferred to 12ml falcon tube, centrifuged at 10000 rpm for 5min, 4˚C, collected the pellet resuspended in 600 µl of SM buffer and transferred to a sterile 1.5 ml Ependorf tube and twice 600 µl of phenol: chloroform: isoamyl alcohol (25:24:1) was added and thoroughly mix. This was then centrifuged at 12000rpm for 15 min, the upper aqueous layer was transferred in a fresh 1.5ml centrifuged tube, an equal volume of chloroform was added and mixed well, and this was centrifuged 12000 rpm for 20 min at 4˚C. collect the upper aqueous  layer  was  transferred  to  a  new  1.5  ml  Eppendorf tube, add 0.3 M sodium acetate (pH 5.3) and  an  equal  volume  of  isopropyl  alcohol  was  added. This was vortexing and incubated at -80˚C for 10 min, then centrifuged at 12000 rpm for 20 min, supernatant was removed and add 700 µl of 70% ethanol and then centrifuged at 12000rpm for 10 min. Then dried pellet was dissolved in 50 µl of TE buffer (10 mM Tris HCl and 1 mM EDTA [pH 8.0])

All the best. 

@Nisha Rathor , Chances remain always but in small quantity of bacterial DNA that does not effect your main experiment , because you add PEG after doing phage purification (Filteration,ultracentrifugation ,dialysis lowers the bacterial contamination.)

Thanks Pallavali Rojarani

concentrate your lysate first with either vaccum evoporation and or PEG NaCl. then add DNase and RNase, that will eliminate any bacterial contaminant of DNA and RNA. then proceed as normal DNA isolation steps.

As Naveen Chaudhary mentioned, Norgen has a bacteriophage DNA isolation kit now and it's especially for a wide range of bacteriophage DNA, rather than an added protocol like Promega.

Proceed your phage lysate for DNA extraction. Prior to DNA extraction, pre-treat the lysate with DNase and RNase enzymes. After this, add EDTA (20mM), SDS 0.5% and Proteinase K (usually 20-40mg/mL). Incubate this at 65 degrees for 30 mins and then proceed with 2 rounds of Phenol: Chloroform: Isoamyl alcohol and one round of C: I, followed by ethanol precipitation (95%), Incubate this at -20 degrees for 6 hrs and then centrifuge at high speed around 10-12000 rpm. Now decant the supernatant and air dry the pellet and resuspend in 40uL RNase free water and store at -80 degrees.

Dear Pallavali, thanks for your protocol, helped me a lot.