Link to this post | posted 21 May, 2019 17:52
khafer	additional photos…can't figure out how to add multiple attachments! 535Kb

Link to this post | posted 21 May, 2019 17:53
khafer	additional photo 579Kb

Link to this post | posted 21 May, 2019 19:10

dwilliams

I am incredibly interested in this post and would love to know the mechanism by which DNA modification can lead to instability of purified DNA under specific conditions. At the very least, it is an interesting puzzle to solve…. I saw this last year with some smeg phages. I forget the specific ones, but recall that I attributed the smearing to residual nuclease present that refolded in RE buffer based on smears present when RE buffer was added, but not no buffer added controls. This year with Gordonia phages and pre-treating them with PK and SDS, we had OK success with getting DNA. By OK I mean that there wasn't the smearing of extracted DNA, but addition of enzyme didn't result in production of a digested genome. Because I don't know whether the enzyme sites were present in the genomes analyzed, I don't know whether enzyme cleavage failed or not. I look in notebooks tomorrow and see if I can find anything. dw

Link to this post | posted 23 May, 2019 05:03

ejyoon

We saw some smearing in our gels. This was our first year so we did not know what to expect and believed the smearing to be due to human or equipment error (and we are not ruling that out). I've attached an image of a gel that shows faint smearing throughout the cut lanes (not much uncut DNA present in the first lane unfortunately). Fortunately, we were able to get sequencing back for our phages (the attached gel image is from one of those sequenced phages). 257Kb

Link to this post | posted 23 May, 2019 17:32

Pollenz

We have worked in Gordonia r. and M. foliorum the past few years. Typically, the uncut DNA from isolated that have high concentration (> 200ng/ul) runs with a high mol mass band, but does show some amount of smear (see enclosed example). The gels sometimes show a double band or gap in the high mol mass DNA. If we have low yield samples (<50ng/ul), they sometimes do not run as a high mol mass band and appear smeared but its hard to tell if that's because the concentration is already low and was degraded during the isolation. RS Pollenz 1.41Mb

Link to this post | posted 23 May, 2019 21:09

ettingera

Hi, Debbie- After switching to the M. foliorum phage from M. smegmatis phage our DNA has been more smeary in general, and doesn't consistently cut as cleanly. In our Genetics/Bioinformatics part of our bio series we run ~300 ng, uncut DNA next to Sal I cut DNA prior to cloning the fragments into pBluescript plasmids, and then sequence analysis of the interesting clones. Gels were run overnight at 25V. We do NOT put Restriction Digest Buffer in our Uncut samples. The uncut DNA is mixed with sterile water and 6X load dye (no SDS, but contains EDTA) before loading, and this has worked well to protect DNA from degrading. Our cloned phage DNA fragments this semester matched to about 50% EE and/or EG phage, and 50 % M. foliorum bacterial fragments. Why so much bacterial DNA ligated into our plasmid? My summer project is to try another enzyme, Nsp1, on these phage since it appears to cut previously sequenced M. foliorum phage more consistently than Sal I. Results later…. None of the smeary phage DNA samples, shown in gels attached, was successfully ligated into the plasmid we use, pBluescript. It ran smeary, and didn't cut cleanly….Interesting! -Ann-Scott 2.05Mb

Link to this post | posted 29 May, 2019 20:16

c.sunnen

I thought I'd chime in, as this is something we've worked hard at to eliminate. A few things to note: 1) We purchase nuclease-free water for suspending our DNA as we've had issues in the past with our smeg phages; we would get beautiful bands with fresh DNA, but even our uncut would smear if left over night, or when thawed. This was mostly remedied by using the nuclease-free water. 2) We run TAE gels (not TBE). They're much cleaner in our hands. 3) We do not use the Wizard kit AT ALL for DNA isolation. We have been using the ZnCl protocol for 2 years now. 4) We isolated M. foliorum phages for the first time this year (smeg previously). After following some threads/discussion about smearing being a common issue with this host, I tried to get ahead of the game by adapting our ZnCl DNA isolation protocol to include the EDTA and ProtK steps. I've attached it here (dated 201, as we had really good results, and tested the effect of water vs. buffer on smearing. While the enzymes didn't work great, the DNA was clean and we didn't see any smearing. In the next post, I've shared pictures from our gels. 16Kb

Link to this post | posted 29 May, 2019 20:18
c.sunnen	On the large gel (attached, 1.2% agarose), after the ladder, each sample set is loaded as: water uncut, NspI, SacII. On the small gel (next post, 2% agarose), the sets are loaded as: buffer uncut, HaeIII. So large gel lanes 1-3 and small gel lanes 1-2 are the same phage. 264Kb

Link to this post | posted 29 May, 2019 20:19
c.sunnen	On the large gel (previous post, 1.2% agarose), after the ladder, each sample set is loaded as: water uncut, NspI, SacII. On the small gel (attached, 2% agarose), the sets are loaded as: buffer uncut, HaeIII. So large gel lanes 1-3 and small gel lanes 1-2 are the same phage. 264Kb

Link to this post | posted 30 May, 2019 18:21
mastropm	We have had the same issues with streaky samples. We changed procedures and were able to increase yield of our samples. We still had some smearing and sent ours anyway and only had one sample pass QC for sequencing. Edited 30 May, 2019 18:28 68Kb