SEA-PHAGES | DNA Smear

Link to this post \| posted 20 May, 2019 19:33
debbie	Dear SEA-PHAGES faculty, Have you see phage DNA appear as a smear on an agarose gel? There are particular instances in which DNA modification can lead to degradation in the gel buffers, such that even undigested genomic DNA appears as a smear. It’s possible that you’ve observed it but not necessarily submitted the DNA for sequencing. We would like to hear whether or not you’ve seen this. We’ve opened up a Forum at seaphagesdb.org, and would like to encourage you to indicate with which phages you saw this (with pictures if you have them) or point to a phage pages on phagesdb.org if the data are posted there. But we’d also like it if you could post a note saying that you have never seen this, if that is the case. If you have further questions, don’t hesitate to contact me. Thanks! debbie

Link to this post | posted 20 May, 2019 19:33

Dear SEA-PHAGES faculty,

Have you see phage DNA appear as a smear on an agarose gel? There are particular instances in which DNA modification can lead to degradation in the gel buffers, such that even undigested genomic DNA appears as a smear. It’s possible that you’ve observed it but not necessarily submitted the DNA for sequencing.

We would like to hear whether or not you’ve seen this. We’ve opened up a Forum at seaphagesdb.org, and would like to encourage you to indicate with which phages you saw this (with pictures if you have them) or point to a phage pages on phagesdb.org if the data are posted there.

But we’d also like it if you could post a note saying that you have never seen this, if that is the case.

If you have further questions, don’t hesitate to contact me.

Thanks!
debbie

Link to this post \| posted 20 May, 2019 21:13
scaruso	We certainly had problems with smears in the past. Most of the time, I have attributed it to user error, and we have switched to adding EDTA and proteinase K to our protocol. It has nearly, but not completely eliminated the issue. I think the few that remain are a mixture mostly of students that are incautious, and a few that are something else. We have phages that simply refuse to give us pretty DNA (the Bacillus phi29-like podos are a reliable example) unless we use phenol chloroform despite very high titers. That's consistent over several years among many students. We have to wonder if there is something interesting going on with their DNA or capsid, or a packaged enzyme that is being denatured by the phenol but not by the standard isolation. Then, this year, we had several, five I think, Streptomyces phages that were not cut at all when tested by every enzyme I could get my hands on (I think I had ten or so). These were enzymes that were consistently cutting other phages' DNA, so we knew they worked. So, not a smear, but modified DNA? Seems like a possibility. Steve

Link to this post | posted 20 May, 2019 21:13

scaruso

We certainly had problems with smears in the past. Most of the time, I have attributed it to user error, and we have switched to adding EDTA and proteinase K to our protocol. It has nearly, but not completely eliminated the issue. I think the few that remain are a mixture mostly of students that are incautious, and a few that are something else.

We have phages that simply refuse to give us pretty DNA (the Bacillus phi29-like podos are a reliable example) unless we use phenol chloroform despite very high titers. That's consistent over several years among many students. We have to wonder if there is something interesting going on with their DNA or capsid, or a packaged enzyme that is being denatured by the phenol but not by the standard isolation.

Then, this year, we had several, five I think, Streptomyces phages that were not cut at all when tested by every enzyme I could get my hands on (I think I had ten or so). These were enzymes that were consistently cutting other phages' DNA, so we knew they worked. So, not a smear, but modified DNA? Seems like a possibility.

Steve

Link to this post \| posted 20 May, 2019 21:42
wynn	It has been a while since I taught SEA-PHAGES, but I think some of my students' phage DNA appeared smeary on their gels. I did a quick check of their final posters and found the attached image, which shows some smearing of uncut DNA in lane 3. Is this the kind of smearing you mean? Let me know if you need a higher resolution image, and I can try to track it down from the lab notebook. If it's of interest to you, the phage is Scootypjr (phage page: https://phagesdb.org/phages/Scootypjr/). The student wasn't able to extract enough DNA to send for sequencing, but there is an archived lysate available. 335Kb

Link to this post | posted 20 May, 2019 21:42

wynn

It has been a while since I taught SEA-PHAGES, but I think some of my students' phage DNA appeared smeary on their gels. I did a quick check of their final posters and found the attached image, which shows some smearing of uncut DNA in lane 3. Is this the kind of smearing you mean? Let me know if you need a higher resolution image, and I can try to track it down from the lab notebook.

If it's of interest to you, the phage is Scootypjr (phage page: https://phagesdb.org/phages/Scootypjr/). The student wasn't able to extract enough DNA to send for sequencing, but there is an archived lysate available.

Link to this post \| posted 20 May, 2019 21:43
robward	Hi Debbie, Yes, we noticed that this year for sure. It was clearly the case for the Gordonia phages Square and Rosemary, and I think it was happening in phage Grass. I will put the figures together and send them to you as soon as I can. I have to look back to last year, but its likely we saw it then as well. Rob

Link to this post \| posted 20 May, 2019 22:29
robward	Hi Debbie, Here is the example of smeared DNA for Square and Rosemary. Rob 4.66Mb

Link to this post \| posted 20 May, 2019 23:06
amandabbraley	We've had quite a bit of smeary DNA from our M. foliorum phages over the last two years. We adopted the Proteinase K/EDTA steps and investigated a few other modifications, including changing some of the temperatures used during the nuclease steps. Our thoughts were that the capsids of some phages were very "unstable" and didn't protect the phage DNA during the extraction protocol. I've attached the poster our students took to the SEA SYMPOSIUM last summer about this issue. 1.43Mb

Link to this post | posted 20 May, 2019 23:06

amandabbraley

We've had quite a bit of smeary DNA from our M. foliorum phages over the last two years. We adopted the Proteinase K/EDTA steps and investigated a few other modifications, including changing some of the temperatures used during the nuclease steps. Our thoughts were that the capsids of some phages were very "unstable" and didn't protect the phage DNA during the extraction protocol. I've attached the poster our students took to the SEA SYMPOSIUM last summer about this issue.

Link to this post \| posted 20 May, 2019 23:07
amandabbraley	I also just grabbed some screenshots from student posters this semester to show the range of results we see with M. foliorum phages. There are 8 different phages shown here. 381Kb

Link to this post \| posted 20 May, 2019 23:08
mssaha	Hi Debbie, We have been working with M. aichiense phages. We have not generally seen smears; we have seen virtually nothing. We have had a terrible time with low titers and therefore getting sufficient DNA. We have had to use a large liquid culture and PEG precipitation to get even a microgram of nanogram amounts of DNA.We have seen some very low molecular weight smears with some of these including HerbertWM (which we rarely to never have seen before). Margaret

Link to this post | posted 20 May, 2019 23:08

mssaha

Hi Debbie, We have been working with M. aichiense phages. We have not generally seen smears; we have seen virtually nothing. We have had a terrible time with low titers and therefore getting sufficient DNA. We have had to use a large liquid culture and PEG precipitation to get even a microgram of nanogram amounts of DNA.We have seen some very low molecular weight smears with some of these including HerbertWM (which we rarely to never have seen before). Margaret

Link to this post \| posted 21 May, 2019 00:43
smolloy123	Hi Debbie, We have this problem every year with certain phage (ironically one phage's name was faintshadow!). Last year, we struggled with a phage called Eden, a cluster EB phage. We were never able to successfully do a restriction digest on it but Dan was able to sequence it. Cheers Sally

Link to this post \| posted 21 May, 2019 17:50
khafer	We had problems with smeary DNA for the first time this year. We used the host Streptomyces griseofuscus, which we also used last year without problems. Eventually I found that the uncut DNA looked fine if I ran it out right after isolation, but even one day in the freezer and the DNA looked smeary. Sorry I don't have gels directly showing this but I have some gels showing non-smeary and smeary DNA. We eventually changed DNA preps and sent the DNA we got for sequencing. The good news is all the samples worked fine for sequencing. Kathy 738Kb

Link to this post | posted 21 May, 2019 17:50

khafer

We had problems with smeary DNA for the first time this year. We used the host Streptomyces griseofuscus, which we also used last year without problems. Eventually I found that the uncut DNA looked fine if I ran it out right after isolation, but even one day in the freezer and the DNA looked smeary. Sorry I don't have gels directly showing this but I have some gels showing non-smeary and smeary DNA. We eventually changed DNA preps and sent the DNA we got for sequencing. The good news is all the samples worked fine for sequencing.

Kathy

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