SEA-PHAGES | All posts created by viknesh

Link to this post \| posted 16 Oct, 2017 17:44
viknesh	mdgainey Hi all, hope your semester is going well. We are currently working with phages isolated from Microbacterium foliorum… 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.

Link to this post | posted 16 Oct, 2017 17:44

mdgainey
Hi all, hope your semester is going well. We are currently working with phages isolated from Microbacterium foliorum…

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
viknesh	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. 87Kb

Posted in: General Message Board → Bacteriophage Microbiologist Position

Link to this post \| posted 25 Sep, 2017 13:27
viknesh	Pollenz I am unable to find the 200-400 mesh carbon-formvar coated copper grids as specified in the ordering guide and listed as item #1813. Is there a typo or can someone help me to purchase the correct item as I have no experience with these materials. 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 25 Sep, 2017 13:27

viknesh

Pollenz
I am unable to find the 200-400 mesh carbon-formvar coated copper grids as specified in the ordering guide and listed as item #1813. Is there a typo or can someone help me to purchase the correct item as I have no experience with these materials.

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

Posted in: General Message Board → EM GRIDS PURCHASE ITEM 1813 not showing up

Link to this post \| posted 17 Nov, 2016 20:58
viknesh	Hi Stephanie, I would be great if you could upload it here. Vic Edited 17 Nov, 2016 20:58

Posted in: Phage Discovery/Isolation → Cluster Specific Primers

Link to this post \| posted 16 Nov, 2016 16:21
viknesh	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? 140Kb

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 11 Nov, 2016 15:08
viknesh	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

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 09 Nov, 2016 21:24
viknesh	Rodney King Has anyone experienced degradation of DNA isolated by the new protocol? We tried a brand new DNA isolation kit and brand new enzymes and still see degradation. Never experienced this problem before. The fact that the DNA disappears only in the presence of restriction enzyme buffer indicates DNase is not being thoroughly removed. 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 Edited 10 Nov, 2016 00:28

Link to this post | posted 09 Nov, 2016 21:24

viknesh

Rodney King
Has anyone experienced degradation of DNA isolated by the new protocol? We tried a brand new DNA isolation kit and brand new enzymes and still see degradation. Never experienced this problem before. The fact that the DNA disappears only in the presence of restriction enzyme buffer indicates DNase is not being thoroughly removed.

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

Edited 10 Nov, 2016 00:28

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 08 Nov, 2016 18:38
viknesh	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

Link to this post | posted 08 Nov, 2016 18:38

viknesh

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

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 03 Nov, 2016 18:35
viknesh	Hi Rodney, I can't say I've seen this, and have not yet heard from anyone with this problem. Perhaps someone who has will chime in. In the meanwhile, would you share a gel where you see the degradation only in the presence of restriction enzyme buffer? I assume the buffer isn't contaminated with DNAse, and that you are using brand new buffer when you used the new enzymes? Vic Edited 03 Nov, 2016 18:36

Link to this post | posted 03 Nov, 2016 18:35

viknesh

Hi Rodney,

I can't say I've seen this, and have not yet heard from anyone with this problem. Perhaps someone who has will chime in.
In the meanwhile, would you share a gel where you see the degradation only in the presence of restriction enzyme buffer? I assume the buffer isn't contaminated with DNAse, and that you are using brand new buffer when you used the new enzymes?

Vic

Edited 03 Nov, 2016 18:36

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 02 Nov, 2016 15:51
viknesh	Emily, do you have a lysate of arthrobacter phage that you use as a control? Has this also stopped producing plaques? Vic

Posted in: Arthrobacter → Cell growth issues

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