SEA-PHAGES | Restriction Enzyme Digests

Link to this post \| posted 14 Oct, 2017 12:04
mdgainey	Hi all, hope your semester is going well. We are currently working with phages isolated from Microbacterium foliorum. I have thus far observed lower DNA yields with these phages than with phages from Mycobacterium at comparable titers. I have been able to get decent yields by peg precip/pelleting, but have noticed for about half of my phages that I am likely precipitating a nuclease of some type (in preps done without DNase/RNase treatment that are not for sequencing). Hopefully an sds/edta/protK-55degree incubation before the Wizard kit will remedy this problem. I also think that some of these phages are less stable at 4 degrees than the phages from Mycobacterium, so perhaps preps from very fresh stocks may be best? Has anyone else observed this? I also wanted to perhaps make an effort to standardize the restriction enzymes digests that we are using and uploading to phagesdb.org. Many of the Microbacterium phages on phagesdb do not have digest pics, and the ones that do often have a single digest shown, but I am not sure what enzyme has been used. In the guide HaeIII, NspI, and XceI are suggested. How are these digest looking for those that have tried these enzymes, and what conditions are you using to run your gels. I am assuming HaeIII is done on a very high percentage agarose gel and would love any tips that anyone has about running these. Thus far my phages fall into two groups, ones that are cut by the traditional enzymes (Bam,cal,eco,hae, hind) and those that are only cut by HaeIII. Lastly, I have found the phageenzyme tool to be very useful in the past for preliminary cluster assignment of my phages and would like to continue using it for the phages isolated from Microbacterium but many of the sequenced Microbacterium phages are not yet popping up when I use this tool. Is the tool updated periodically, or should I request to have certain phages added?

Link to this post | posted 14 Oct, 2017 12:04

Hi all, hope your semester is going well. We are currently working with phages isolated from Microbacterium foliorum. I have thus far observed lower DNA yields with these phages than with phages from Mycobacterium at comparable titers. I have been able to get decent yields by peg precip/pelleting, but have noticed for about half of my phages that I am likely precipitating a nuclease of some type (in preps done without DNase/RNase treatment that are not for sequencing). Hopefully an sds/edta/protK-55degree incubation before the Wizard kit will remedy this problem.
I also think that some of these phages are less stable at 4 degrees than the phages from Mycobacterium, so perhaps preps from very fresh stocks may be best? Has anyone else observed this?
I also wanted to perhaps make an effort to standardize the restriction enzymes digests that we are using and uploading to phagesdb.org. Many of the Microbacterium phages on phagesdb do not have digest pics, and the ones that do often have a single digest shown, but I am not sure what enzyme has been used. In the guide HaeIII, NspI, and XceI are suggested. How are these digest looking for those that have tried these enzymes, and what conditions are you using to run your gels. I am assuming HaeIII is done on a very high percentage agarose gel and would love any tips that anyone has about running these. Thus far my phages fall into two groups, ones that are cut by the traditional enzymes (Bam,cal,eco,hae, hind) and those that are only cut by HaeIII.
Lastly, I have found the phageenzyme tool to be very useful in the past for preliminary cluster assignment of my phages and would like to continue using it for the phages isolated from Microbacterium but many of the sequenced Microbacterium phages are not yet popping up when I use this tool. Is the tool updated periodically, or should I request to have certain phages added?

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

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 17 Oct, 2017 14:18
mdgainey	Thanks Vic, I did not include the nucleases in this round of preps bc I have already run into an unstable capsid (DNA fully degraded before RE digest). After precipitation and prep, the DNA looks fine for the other phages that I have not added nucleases to, but as you said after adding buffer and placing at 37 degrees I see degradation. It seems that the phages that are cut by the traditional enzymes are the ones that I am seeing the degradation with. I will know next week how the rest of the class's preps look with the nuclease inactivation step added. I have never had good luck getting rid of the nucleases with the resin from the Wizard kit, and normally do the nuclease inactivation step with every prep that we do that nucleases have been added. I will have the students do an NspI digest and post that to phages db. Maria

Link to this post | posted 17 Oct, 2017 14:18

mdgainey

Thanks Vic,
I did not include the nucleases in this round of preps bc I have already run into an unstable capsid (DNA fully degraded before RE digest). After precipitation and prep, the DNA looks fine for the other phages that I have not added nucleases to, but as you said after adding buffer and placing at 37 degrees I see degradation. It seems that the phages that are cut by the traditional enzymes are the ones that I am seeing the degradation with. I will know next week how the rest of the class's preps look with the nuclease inactivation step added. I have never had good luck getting rid of the nucleases with the resin from the Wizard kit, and normally do the nuclease inactivation step with every prep that we do that nucleases have been added. I will have the students do an NspI digest and post that to phages db.

Maria

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Restriction Enzyme Digests