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Recent Activity
All posts created by viknesh
Link to this post | posted 07 Jul, 2018 14:53 | |
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asprenkle Hi Amy, The app is down at the moment. When you check-in at UMBC, we'll tell you how to go about sample collection without using the app. See you soon! |
Posted in: SEA-PHAGES App → a work in progress
Link to this post | posted 10 Nov, 2017 18:24 | |
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Adaptive Phage Therapeutics (APT) is currently in need of experienced bacteriophage microbiologists to join our team. Attached to this post is a job description for a BS- or MS-level bacteriophage microbiologist. APT is a clinical-stage pharmaceutical company based in Gaithersburg, Maryland that is focused on developing an effective and personalized bacteriophage therapy in response to the global rise of multi-drug resistant (MDR) bacterial pathogens. Two key components to APT's approach include a large and dynamically growing collection of bacteriophages, or PhageBank™, and an innovative rapid system for matching bacteriophage to patient-specific bacterial infections called the Host Range Quick Test. See links below for more information about Adaptive Phage Therapeutics, Inc.: Company Website: http://www.aphage.com News Articles: 1) http://www.businesswire.com/news/home/20170426006880/en/Adaptive-Phage-Therapeutics---Terminally-Ill-Patient 2) https://www.buzzfeed.com/azeenghorayshi/navy-phage-viruses-for-antibiotics-crisis?utm_term=.gvym0OEXQ#.vdmjdPXNV 3) https://www.nbcnews.com/mach/science/post-antibiotic-apocalypse-can-be-prevented-here-s-how-ncna812576 |
Link to this post | posted 16 Oct, 2017 17:44 | |
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mdgainey Hi Marie, We've done some work with M. foliorum. The yields from the 8 or so preps were similar to what we typically get with mycobacteriophages, which is 60 - 80 ng/ul from 1 ml of a e9 pfu/ml lysate. When we PEG-precipitate phage, the DNA yields increased proportionately with the volume of lysate used. With regard to degraded DNA, we (including several SEA faculty) have noticed that some DNA preps that were isolated using the new isolation protocol (no PEG precipitation) is degraded upon mixing with restriction enzyme buffer. We think this may indicate that some of the nucleases added are coming through the DNA prep, and activated when mixed with the restriction enzyme buffer. This does not seem to happen when PEG precipitation in included, suggesting that the precipitation step is helpful with removing some/most of the nucleases that are added. In your case, where no nucleases are added, its surprising that you still see degraded DNA. Do you see degraded DNA when run on a gel without restriction enzyme buffer? One other possibility we've entertained is that when the Wizard denaturation mix is not properly mixed before use, you might not have full inactivation of nucleases that are added or host bacterial nucleases present in the lysate. You might have to give us more information about how you are performing your DNA isolation, and when and where you are seeing the degraded DNA. As for restriction enzymes, we tested a panel of enzymes and found NspI (also called XceI) is the most diagnostic enzyme. The majority of other enzymes tested did not cut DNA. SacII was able to digest one or two phage DNA preps, and HaeIII, as expected, cut so many times that unless a high percentage gel is used, is not informative. If you search for phage Eleri on pahgesDB, the restriction gel on the web includes several other M. foliorum phages for comparison. |
Posted in: Microbacterium → Restriction Enzyme Digests
Link to this post | posted 10 Oct, 2017 15:17 | |
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Yet another full-time position at a phage therapy company (same company as in the previous post) for a BS/MS with phage experience. Located in Gaithersburg, MD. |
Link to this post | posted 25 Sep, 2017 13:27 | |
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Pollenz The cat # is 01813. The 0 was dropped from the original excel sheet (standard excel formatting), but has now been updated. Were you able to find the item? Vic |
Link to this post | posted 17 Nov, 2016 20:58 | |
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Hi Stephanie, I would be great if you could upload it here. Vic |
Posted in: Phage Discovery/Isolation → Cluster Specific Primers
Link to this post | posted 16 Nov, 2016 16:21 | |
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Dan, here is a representative gel (of ~ 40 gels) from the workshop when faculty used the new DNA isolation protocol. As you can see, there is some amount of smear. Is this what you are receiving for sequencing? |
Link to this post | posted 11 Nov, 2016 15:08 | |
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Hi Laurie, Is this true across the board, or are only 3 students getting low DNA yields? A little more cumbersome, but you could try to concentrate the phage, either using the EM protocol for concentrating phage, or the PEG precipitation protocol. When concentrating, you should spin at 4C. Vic |
Link to this post | posted 09 Nov, 2016 21:24 | |
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Rodney King Hi Rodney, I had a conversation with Steve Caruso today, and he's seen what you've been describing. He thinks it happens when students remove the resin/denaturant without first properly mixing the solution. As a result, the DNase added to the lysate/resin mix may not be fully denatured. Any remaining folded DNase may be inactive during purification because of the presence of EDTA. In the elution (in water), perhaps there isn't sufficient cations to support DNase activity. The addition of the restriction buffer, however, bring in cations that might support DNase activity. Thoughts? Vic |
Link to this post | posted 08 Nov, 2016 18:38 | |
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Hi Cathy, Are you getting low yields across the board or just for a number of students/samples? Typically, one can expect a yield of ~ 40ng/ul or higher from a 10^9 pfu/ml lysate when prepping using the protocol 9.1. If a lysate yields low DNA, there is always the option to concentrate the lysate by centrifugation (see PEG precipitation, or the TEM protocol). Regardless of the protocol you use for concentrating phage by centrifugation, it will be important that you spin at 4C. If you are seeing low yields across the board, perhaps its because of a bad reagent. Vic |