I am now working on the bacteria Burkholderia Pseudomallei strain K96243 and the phage I isolated from the soil sample.
I am trying to prepare a high titre lysate for the use in protein extraction later.
Here is the protocol I used:
1. Incubate 10ml of the bacterial culture (+100ul overnight bacterial culture) until OD reach 0.6
2. Add in 100ul of phage stock and continue to incubate
3. After cell lysis is observed, add in few drops of chloroform and centrifuge (10000rpm/ 30min/ 20C)
*while waiting for centrifugation, i put the 20% PEG-8000 solution on ice
4. Syringe filter the supernatant using 0.20um syringe filter
5.Mix 2ml of 20% cold PEG-8000 solution with 8ml of filtered supernatant
*here i tried 20% PEG-8000 alone & 20% PEG-8000/ 2.5M NaCl -- but both show no pellet
*for 20% PEG-8000 alone- 20g PEG-8000 + 80ml dH2O and heat the solution at 90C, then filter it (0.45 um syringe filter)
*for 20% PEG-8000/2.5M NaCl- 20g PEG-8000 + 14.61g NaCl + 80ml dH2O
6. Put the tube on ice for 1hr
7. Centrifuge (10000rpm/15min/4C)
But no pellet was formed after centrifugation
Is there any mistake I made? or any other method I can try on?
Kindly advised. Thanks.
May i know how, long you took for whole process ? or can you mention timings of each process,
Too short timing may effect on centrifugation result
There are two possibilities to explain your failure to see a pellet:
1) The phage cannot be precipitated under the conditions you tried
2) There is too little phage for you to see the pellet, if any. Did you try to titer your purportedly "high titer lysate ? A quick calculation shows that, for a phage of the size of lambda (~60 MDa), it takes 10exp10 phage particles to get 1 µg of material (total DNA + protein)
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