Link to this post | posted 14 Nov, 2019 03:22

c.sunnen

Hi! My first suggestion is to digest longer; we typically do ours for at least an hour at 37deg or overnight at room temp. Good news is your DNA looks nice! Some smearing, but mostly intact. If you're uncertain if your enzyme is any good, you can try digesting some lambda DNA (provided you have some on hand). I hope this is helpful! Good luck! Nikki P.S. If you're using the protocol I've posted above, there have been a few edits, though it should still work in its current form.

Link to this post | posted 12 Jul, 2019 15:29

c.sunnen

I had something similar in Mashley (EG). Genes 42 and 43 were orphams, the result of a frameshift that split a gene in approximately half. Interestingly, they split a "membrane protein" into two "membrane protein" proteins! Both orphams retained several TM domains! So they both got called that way. In your case, I think calling them both NKF genes is probably appropriate (unless there is a function you can attribute to either/both combined). Without bench data to suggest otherwise, as long as it's got coding potential, it's probably safer to assume it does get made, even if it's non-functional.

Link to this post | posted 11 Jul, 2019 21:12

c.sunnen

Hi! I am QC-ing EE phage Vanisius, and have some good HHPred hits to "tail fiber" for gp 15 (see screenshots in first slide of attached PPT). Most are calling this pham simply a minor tail protein, but while it's in the right place, it's only ~550bp. What's the expected size for a tail fiber? Is this best called NKF, minor tail, or tail fiber? In a related question, gp 9 has some good HHPred evidence for a minor tail protein of some kind (see the 2nd slide in attachment), but is less than 400bp long. It is immediately downstream of the major tail protein and immediately upstream of the tail assembly chaperones. I want to confirm that this is best left as NKF? Thanks! 156Kb

Link to this post | posted 30 May, 2019 19:28
c.sunnen	Matt, Did you use our protocol? Your gel looks similar to ours. We also had some issues with sequencing - turns out we had some bacterial DNA contamination - but we assumed that was user error (asking the TAs to add the nuclease instead of doing it ourselves). We're actually going to test this over the summer. Otherwise, our DNA seemed pretty clean. Nikki

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 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 20:16

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 27 Mar, 2019 16:32

c.sunnen

Hi! So tail assembly chaperone has been called a lot for the two genes immediately upstream of tapemeasure in the EA1 phages that we are comparing to, but I am finding no evidence for this, other than synteny, in our EA1 phage, Shee. I can't find a slippery sequence (none of these phages seem to have annotated one, either), and it seems that these phams are only found in EA cluster (and sub-cluster) phages. Is there some compelling evidence I'm missing, or is this one of those cases of perpetuating a mistake? I know that usually, we try NOT to call tail assembly chaperone unless we can annotate the frameshift. Should these actually be NKF? Thanks! Nikki

Link to this post | posted 30 Jan, 2019 18:16

c.sunnen

Update. I looked into this a little further, and took Claire's lead. I included both genes, but the one with the most homology (gp 73, from aa 57-169) was declared "immunity repressor, truncated" and gp 74 (only matching aa 1-54) got called "Hypothetical Protein." I figured the immunity repressor part of it was too small to give it that function, but that it still should be called as a gene. This should look similar to Kalb97 and Pistacio, now. It will keep both genes, including the overlap, but only the one that still belongs to the immunity repressor pham will get the "truncated" label, whereas the other is NKF.

Link to this post | posted 29 Jan, 2019 15:13

c.sunnen

Related question. I'm QC-ing the A3 phage Noella. There are two genes, largely overlapping, both with hits to immunity repressor, but both clearly truncated (the result of either a deletion or an insertion). This is consistent with the clear plaque morphology of the phage, as noted by the annotators. But I hesitate to call both genes "immunity repressor, truncated" since they're so overlapping. Should I call the first one (gp 74, that gets 1:1 alignments for the first 54 amino acids), but delete the second one (gp 73, aligning amino acids 57-169)? Or should I keep them both, but label the second, overlapping gene as "hypothetical protein" and add info to the Discovery Notes? The overlap is HUGE at ~280bp. I've included a DNAMaster screenshot. Thanks for all your input! Nikki 225Kb