Hello everyone,
I have to isolate DNA from Brucella phage , but before that, I have to concentrate the phage using PEG 8000. I have got protocol for concentrating the phage using PEG and TM buffer but the composition, concentration and pH of TM buffer was not clear . So, please suggest the composition.
Thank you
Just to aid quickly: There are some protocols out in the wild I don't know could be of help for you to overcome your problem...(not knowing about your specific demand - with regard to pH and concentration... BUT: Cf.: https://www.researchgate.net/post/How-could-I-maintain-store-bacteriophages-for-a-long-time ------------------------------------------------------------------------------------TM buffer (50 mM Tris pH 7.5, 10 mM MgCl2)(source – Subscription required: https://journals.aps.org/prl/abstract/10.1103/PhysRevLett.106.028102 ) or: ==> ‚TM buffer is T buffer supplemented with 5 mM MgCl2‘: 'T buffer consists of 20 mM Tris/HCl (pH 7.4) and 5 mM 2-mercaptoethanol. TM buffer is T buffer supplemented with 5 mM MgCl2.'YONESAKI et al,
Purification and some of the functions of the products of bacteriophage T4 recombination genes, uvs X and uvs Y; Eur. J. Biochem. 148, 127-134 (1985); open access source:
https://febs.onlinelibrary.wiley.com/doi/full/10.1111/j.1432-1033.1985.tb08816.x or also:
Article The effect of genome length on ejection forces in bacteriophage lambda
for direct article access via Elsevier:
https:__//www.sciencedirect.com/science/article/pii/S0042682206000067 (before using the link in your browser delete the UNDERSCORE characters in between https: __ and //!):
(QUOTE:) Materials and methodsPhages λb221cI26 (λb221) and λcI60 were extracted from single plaques, grown in 3 L cultures of E. coli C600 cells and purified by PEG precipitation, differential sedimentation, and equilibrium CsCl gradients, resulting in ∼1013 infectious particles. After purification, phages were dialyzed twice against a 500-fold greater volume of TM buffer (50 mM Tris, 10 mM MgSO4, pH 7.4) ( and more mentions of TM within Materials and Methods).Good luck!(NB: Searching your primary keywords | TM buffer AND bacteriophage* | might yield a lot of possible protocol sources (:-)) )
...and here is another recipe. So yes, as Wolfgang said, there are different protocols around. But from my experience, 10 mM Mg2+ is often the standard and my suspicion is that the Ca2+ is not that important.
TM buffer contains 10 mM MgSO4, 10 mM TRIS and 5 μM CaCl2 with distilled water. Adjust the pH to 7.4 with HCl.
The exact recipe doesn't really matter, it should be buffered (10mM-50mM Tris are all fine) and normally it has some Mg ion (10mM is standard). Ca ion is useful for some phages but not needed for most, so it depends upon the phage whether you need it. It never hurts though.
Bactriophage means a virus that replicate in a bacterium T M prepared accoding different protocols according to the experience among them these conistutence: 50mMTris buffer Ph 7.5,10mMgcl2. ,10mMgso4while 10mMg2+is often standard so moe details accoding to each Lab.
Thank you everyone for your suggestions.
@ Salemeh Barhoom, we can use either 10 m M Mgcl2 or MgSo4?I think both of them are not needed? As suggested in earlier discussion only Mg ions are important. I made TM buffer as follows: 302 mg TRIS base and 123.24 mg MgSO4 heptahydrate and dissolved in 30 ml autoclaved distilled water . adjusted ph to 7.5 and then make up the volume to 50 ml . so finally I used 50 mM Tris, 10 mm MgSO4.7H2O. Then used this for phage concentration using PEG followed by DNA isolation using qiagen QIAmp kit but got v. less DNA. Can any body help in this regard.
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