I am working on bactreiophage- I am trying to purify bacteriophage DNA for sequencing using phenol. When I run it still there is smear even when I increase the amount of DNase and RNase.
If you are using a next-generation method (e.g., Illumina) for sequencing, a small amount of bacterial DNA in the sample is acceptable. Otherwise, I would recommend purifying your phages prior to DNA extraction. To obtain a reasonably high concentration of reasonably pure phage DNA, I personally (1) grow phages to a high titer, (2) spin down the lysate, (3) PEG precipitate the supernatant, (4) lyse the phage virions with EDTA and SDS, and then (5) extract phage DNA with phenol chloroform. This has always worked well for me.
thank you very much
for precipitation, is it better if I add solid PEG or making a solution? to lyse the phage virion what volume of EDTA and SDS should I use ?
Here is my protocol.
PEG precipitation
• PEG solution: 20% (wt/v) of PEG 8000/2.5M NaCl
• STE solution: 1mL 1M Tris, 0.2mL 0.5M EDTA, 2mL 5M NaCl, fill to 100mL with de-ionized water.
1) Produce a high-titer phage lysate; spin down and/or pass through 0.44µm filter to remove bacteria and debris.
2) In a 50-ml Falcon tube, add one volume of 20% PEG/2.5M NaCl to four volumes of high-titer phage lysate supernatant, invert several times, and chill on ice for one hour. To obtain a high phage DNA concentration, use a minimum of 10 ml phage lysate (2.5 ml PEG solution).
3) Spin down tube(s) for 20 minutes at 15k RCF or 60 minutes at 4.5k RCF. Upon pouring off the supernatant, a light pellet should be visible at the bottom of the tube (note: it will not be anywhere as noticeable as a bacterial pellet). Carefully remove as much of the supernatant as possible without losing the phage pellet.
4) Add 1 ml STE solution to the tube and fully re-suspend the phage pellet via vortexing and pipetting up and down. Transfer this volume to a microfuge tube.
5) Spin down in a microfuge for 10 minutes at 14k RCF. Retain 1 ml of supernatant – this contains your purified phage.
DNA extraction
1) To your precipitated phage tubes, add 100 µl of 0.5M EDTA (final concentration should be ~50 mM; 1:10 dilution) and 100 µl of 10% (wt/v) SDS (final concentration should be ~1%; 1:10 dilution). Invert tubes several times.
2) Add 250 µl of standard phenol:chloroform:isoamyl alochol to each tube, vortex thoroughly, spin down for four minutes at 10k RCF, and withdraw ~700 µl of supernatant to a fresh tube. Be careful to NOT disturb the phenol layer – this will yield a low-quality DNA sample.
3) To each tube, add 630 µl pure isopropanol and 70 µl of 5M NaCl and gently invert (do not vortex). Most of the time, you should see a visible DNA precipitate form. Spin down for 10 minutes at 10k RCF.
4) Pour off supernatants and remove remaining volume with a micropipette tip. You should see a DNA pellet in the bottom of the tube at this point; if not, something is probably wrong. Add 500 µl of 70% ethanol (note: the DNA is very stable in 70% ethanol and can be refrigerated indefinitely at this stage with no significant loss of DNA concentration or quality).
5) Spin down tubes for 5 minutes at 10k RCF, pour off supernatant, and remove as much ethanol as possible with a micropipette. Open the tubes and allow the pellet to air dry until all remaining ethanol has evaporated.
6) Resuspend DNA pellets in 30-50 µl DNAse-free water by pipetting up and down and flicking the tube (do not vortex).
Following phenol/chloroform extraction, CsCl2 equilibrium density gradient centrifugation will generate a purified concentrated band of phage DNA.
:-) forgive me: CsCl is more than sufficient; nobody likes too much chorine in a salt from the first group of the periodic system of elements. :-)
I have very fast and easy standardized method for phage purification before DNA isolation
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 to supernatant 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 and you can use this mixture for following protocols
a)TEM
b)DNA sequencing
c)In vivo experiments
Follow below easy steps , you will not even require dialysis or cscl gradient protocol
1.Centrifuge 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 and you can use this mixture for following protocols
a)TEM
b)DNA sequencing
c)In vivo experiments
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