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Purpose: Use three methods to get rid of nucleic acid of E. coli, organelles, cell debris, chemicals, etc,which produced in the course of production, purified MS2 bacteriophage, and compared the advantages and disadvantages of three methods; Removed of nucleic acids in four ways ,making MS2 bacteriophage to be internal control, and compare advantages and disadvantages of four methods.Methods: MS2 bacteriophage contains a lot of contaminations was purified by three methods, ultracentrifugation, chloroform extraction method, molecular sieve method, comparative advantages and disadvantages of three methods; Respectively application of enzymatic digestion,infinite dilution and dialysis comprehensive, ultracentrifugation , Millipore pillars method to remove nucleic acids,those mixed with the MS2 bacteriophage.Results: MS2 phage purified by three different ways. Ultracentrifugation: Electrophoresis analysis diagram shows ,the sample is high brightness of band ,but tailing phenomenon, and electron microscope analysis, the background is not good, containing a lot of contamination. Chloroform Method: Electrophoresis analysis diagram shows the sample is high brightness of band, no tail phenomenon, and electron microscope analysis, the background is better, does not contain contamination, but SDS-PAGE analysis, the samples have a little of hybrid protein residues. Molecular sieve method: SDS-PAGE analysis the samples without other protein residues, the monomers only 14KD.Using four different methods to remove nucleic acids. Enzyme digestion: Application of four different Concentrations 50U, 25U, 12.5U,and 2.5U, the results is the CT value is similar, that means has the same amount of DNA template. Infinite dilution and dialysis synthesis method: PCR analysis shows,only a slight difference between before and after dialysisthe sample,with the dilution degree increase from 10-2 to 10-7 ,electrophoretic bands are almost the same, and between before and after dialysis samples are also the same, with or without nuclease enzyme is similar, too.Electron microscopy shows after dialysis the sample mixed with a large number of chemical reagents .Ultracentrifugation: PCR product electrophoresis shows the seventh residue after centrifugation precipitated DNA only appeared in 25 cycles, and absorb the effect of DNA, the amount of MS2 phage is still large amount. However, accurate quantification by Real Time PCR, the supernatant precipitation and residual DNA in the phage and almost the same amount, and each time lost approximately 10 times of phage. Millipore column method: electrophoresis shows, PBS Buffer group and Bezonase Buffer group only at 25 cycles when a small amount of DNA template was amplified, Bezonase Buffer is high brightness, and by RT-PCR detection, PBS Buffer group MS2 bacteriophage and the nucleic acid residue is 4 orders of magnitude, while the Bezonase Buffer only 3 orders of magnitude, but Bezonase Buffer is purer than PBS Buffer group ,but both group are better than that did not use this way.Conclusion: Purity of MS2 phage,use trichloromethane extract is better than ultracentrifugation; A great quantity of DNA can be removal when use millipore post,the other is inefficient or worse.
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