SEA-PHAGES | All posts created by mdgainey

Link to this post \| posted 11 Dec, 2017 14:27
mdgainey	Dan Russell mdgainey Thanks for posting this Nick. Dan, I have one podo with an unstable capsid and would like to try to seq/assemble with just an RNase treatment. Dan do you have any basic tips to reduce host DNA contamination in my prep to hopefully achieve a successful assembly without DNase treatment, sounds like it might be fine but if I could stack the deck a bit that would be great. This one grows really well so high titer should not be a problem. Maria Hey Maria, I don't really have any good tips to reduce the host DNA, but my sense is I'd try sequencing it and see what comes out. If the host DNA really is overwhelming, then it's back to the drawing board, but I think there's a decent chance the phage sequence would stand out from the bacterial chunks, and then you'd be done. So I'd just try prepping and sequencing without DNAse and see what comes out, then only worry about reducing the host DNA if that first attempt didn't work. –Dan Thanks Dan, I will give it a go next semester. Maria

Link to this post | posted 11 Dec, 2017 14:27

Dan Russell
mdgainey
Thanks for posting this Nick. Dan, I have one podo with an unstable capsid and would like to try to seq/assemble with just an RNase treatment. Dan do you have any basic tips to reduce host DNA contamination in my prep to hopefully achieve a successful assembly without DNase treatment, sounds like it might be fine but if I could stack the deck a bit that would be great. This one grows really well so high titer should not be a problem.
Maria

Hey Maria,

I don't really have any good tips to reduce the host DNA, but my sense is I'd try sequencing it and see what comes out. If the host DNA really is overwhelming, then it's back to the drawing board, but I think there's a decent chance the phage sequence would stand out from the bacterial chunks, and then you'd be done. So I'd just try prepping and sequencing without DNAse and see what comes out, then only worry about reducing the host DNA if that first attempt didn't work.

–Dan

Thanks Dan, I will give it a go next semester.
Maria

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

Link to this post \| posted 10 Dec, 2017 22:20
mdgainey	Dan Russell Nicholas Edgington Dan: With such great sequence coverage that has been seen from past years, would the contaminating host DNA really cause much of a problem with assembly? Maybe one could just add a bit of RNAse directly to the HTL's? It would be interesting to know if you have ever tried phage genomic sequencing with and without the addition of the DNAse/RNAse cocktail, and compared the assembly results. Hey Nick, We haven't tried that in any real experimental way. We do sometimes get samples with host DNA contamination, and in most cases it's relatively low coverage compared to the phage and thus isn't a problem. On the other hand, we have seen a few samples where the host coverage was high enough to drop the phage coverage to an unusable/undetectable level. So I guess: who knows? Also, has anyone tried side-by-side old protocol versus new protocol to check for yields from each? That would perhaps shed some light. Having a high titer is certainly the most important thing to getting good yield, so bumping up the titer wherever possible is probably the best way to help. –Dan Thanks for posting this Nick. Dan, I have one podo with an unstable capsid and would like to try to seq/assemble with just an RNase treatment. Dan do you have any basic tips to reduce host DNA contamination in my prep to hopefully achieve a successful assembly without DNase treatment, sounds like it might be fine but if I could stack the deck a bit that would be great. This one grows really well so high titer should not be a problem. Maria

Link to this post | posted 10 Dec, 2017 22:20

mdgainey

Dan Russell
Nicholas Edgington
Dan: With such great sequence coverage that has been seen from past years, would the contaminating host DNA really cause much of a problem with assembly? Maybe one could just add a bit of RNAse directly to the HTL's? It would be interesting to know if you have ever tried phage genomic sequencing with and without the addition of the DNAse/RNAse cocktail, and compared the assembly results.

Hey Nick,

We haven't tried that in any real experimental way. We do sometimes get samples with host DNA contamination, and in most cases it's relatively low coverage compared to the phage and thus isn't a problem. On the other hand, we have seen a few samples where the host coverage was high enough to drop the phage coverage to an unusable/undetectable level. So I guess: who knows?

Also, has anyone tried side-by-side old protocol versus new protocol to check for yields from each? That would perhaps shed some light. Having a high titer is certainly the most important thing to getting good yield, so bumping up the titer wherever possible is probably the best way to help.

–Dan

Thanks for posting this Nick. Dan, I have one podo with an unstable capsid and would like to try to seq/assemble with just an RNase treatment. Dan do you have any basic tips to reduce host DNA contamination in my prep to hopefully achieve a successful assembly without DNase treatment, sounds like it might be fine but if I could stack the deck a bit that would be great. This one grows really well so high titer should not be a problem.
Maria

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

Link to this post \| posted 10 Dec, 2017 22:10
mdgainey	Hi all, I posted a couple of weeks ago in the M. foliorum RE digests group, but just saw this thread. I used the Wizard kit for M. smeg phages in the past using the new protocol (however I used some nucleases I had already purchased not the recommended mix) and always had trouble with the nucleases coming through the prep. So basically after nuclease treatment but before the DNA prep I always treat my lysates with sds/protK/edta-55deg for 1 hr (before class and then freeze) and then had the students follow the protocol as normal, and it always worked great. This year I did the same thing but am having issues with about half the M. foliorum and couple of G. Terrae phages we have found after placing them at 37 degrees for digest. More disturbing was that I had the exact same issue when I had the students do nuclease free preps, even with sds/protK/edta treatments before the kit…so I am not sure what is going on. Could it be these phages are bringing a nuclease with them that is even harder to inactivate than the traditional DNases we normally use? I don't think this is a reagent issue as about half of the phages DNA that I put through the preps this year came out looking pristine, and this same procedure has worked like clockwork for me in the past.

Link to this post | posted 10 Dec, 2017 22:10

mdgainey

Hi all, I posted a couple of weeks ago in the M. foliorum RE digests group, but just saw this thread. I used the Wizard kit for M. smeg phages in the past using the new protocol (however I used some nucleases I had already purchased not the recommended mix) and always had trouble with the nucleases coming through the prep. So basically after nuclease treatment but before the DNA prep I always treat my lysates with sds/protK/edta-55deg for 1 hr (before class and then freeze) and then had the students follow the protocol as normal, and it always worked great. This year I did the same thing but am having issues with about half the M. foliorum and couple of G. Terrae phages we have found after placing them at 37 degrees for digest. More disturbing was that I had the exact same issue when I had the students do nuclease free preps, even with sds/protK/edta treatments before the kit…so I am not sure what is going on. Could it be these phages are bringing a nuclease with them that is even harder to inactivate than the traditional DNases we normally use? I don't think this is a reagent issue as about half of the phages DNA that I put through the preps this year came out looking pristine, and this same procedure has worked like clockwork for me in the past.

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

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

Posted in: Microbacterium → 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

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?

Posted in: Microbacterium → Restriction Enzyme Digests

Link to this post \| posted 06 Feb, 2017 16:23
mdgainey	Hi Lee, on a bit of an unrelated note, would you be willing to share your Streptomyces griseus phage hunting protocols with me? I am getting ready to order this host from ATCC. If you have any tips for success that you are willing to share I would be grateful. I have a group of students doing honors projects that I was going to have do some phage hunting in Great Smoky Mountains National park, where we have had 0 success with m. smeg.

Link to this post | posted 06 Feb, 2017 16:23

mdgainey

Hi Lee, on a bit of an unrelated note, would you be willing to share your Streptomyces griseus phage hunting protocols with me? I am getting ready to order this host from ATCC. If you have any tips for success that you are willing to share I would be grateful. I have a group of students doing honors projects that I was going to have do some phage hunting in Great Smoky Mountains National park, where we have had 0 success with m. smeg.

Posted in: Phamerator → PhamDB: Make your own Phamerator databases

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