Link to this post | posted 12 Oct, 2018 17:09

c.sunnen

Vic, This is incredibly helpful - thank-you! And I'm so glad that you pointed out how we can't add the EDTA before the ZnCl (of course, but I didn't catch it!). Also, I didn't see that a version of this protocol was available in the updated instructor's guide, so now we can see how closely our current version matches! We plan to do a "practice" run with our students next week with small volumes of phage lysate (we find that they always mess up the first time they encounter any DNA isolation protocol, and end up wasting a bunch of precious lysate and reagents), so this will be a good opportunity to try the first workflow you suggested. I'll stress that speed is important when adding EDTA, and hope for the best! Clarification question: I know in the instructor's guide protocol you switch to using the Wizard kit at the capsid denaturation step, whereas traditionally (by our ZnCl protocol) we would do this in TES buffer (0.1M Tris-HCl, pH 8, 0.1M EDTA, 0.3% SDS). Since we don't intend to use the Wizard kit at all, would you recommend eliminating the TES step and just proceeding to the potassium acetate precipitation? It seems that if we're resuspending in EDTA instead of TES, the composition would be very similar, and I'm assuming accomplish the same thing: EDTA concentration would be the same, and then SDS would be added with the proteinase K… What %SDS should be added with the protK? Thank-you again! I'll be sure to let you know how it goes! Nikki Edited 12 Oct, 2018 17:39

Link to this post | posted 12 Oct, 2018 14:38

c.sunnen

Vic, Is the suggestion to use ProteinaseK and SDS instead of EDTA, or to use both? This is our first go with M. foliorum. Also, we'll be using the ZnCl method. Have either of these nuclease inhibition steps been tried with the ZnCl method? I'm attaching our current protocol (modified to include an EDTA step; this is the only change from how we did it with smeg last year), in case that's useful. Thanks all! I love this community! Nikki 16Kb

Link to this post | posted 12 Oct, 2018 14:34
c.sunnen	This is the protocol we use, based upon that publication. It worked well for us last year with M. smeg, and we'll be trying it this year with M. foliorum. The EDTA step we just added, in hopes that it will help with some of the degradation issues the foliorum people seem to have had. Sorry it's coming so late, but perhaps it'll still be useful! 16Kb

Link to this post | posted 28 Sep, 2018 16:04
c.sunnen	Hi all! I just wanted to update everyone that making fresh TA (and ensuring that it was perfectly mixed prior to autoclaving), along with using pre-warmed (not cold) plates has fixed all of our issues. Thank-you all! Nikki

Link to this post | posted 14 Sep, 2018 13:41
c.sunnen	Karen, Thanks! This was another of my suspicions; if there was something different about those bottles that got aliquotted first, vs. those that got aliquotted last. The speed of responses has been exceptional, all! I'll let you know how we fair next week with fresh agar, warm plates, and venting while solidifying! Nikki

Link to this post | posted 14 Sep, 2018 12:53

c.sunnen

Thank-you both! These are the kinds of things I'd suspected (and could explain why the morning class is having issues, but the afternoon class is not; plates are generally already pulled out and sitting at room temp for the afternoon class, but not the morning). Interesting side note: We took a couple bottles of TA out of the water bath and let them sit out at room temp. Many hours later, they are still liquid. So I think a new batch of TA is in order anyway! But we'll be sure to use room temp plates, and we'll keep the water bath at 60deg (relatively hot!). Thanks again! Nikki

Link to this post | posted 13 Sep, 2018 18:52

c.sunnen

Hi all! This is the first year we're working with M. foliorum, so we've switched to PYCa from 7H9. We've had issues with TA solidifying in the past, and the solution was to increase our agar concentration. But I'm wondering if there is any further insight/ideas as to what's happening in this particular case. We make individual bottles (~100-200mL) 1x TA and keep them in a water bath at 60deg, so that each pair of students has their own bottle. All bottles are aliquoted from the same stock, right out of the autoclave. Students are instructed to take their bottles out and wait until they can safely handle them (not to hot to pick up), before plating. This is usually on the order of 5-10min. Some of my students are having success plating, and others can't get their TA to solidify; even when left out for 2+ hours! In one case, I had a student plate 12 plates total; her first one never solidified, but the other 11 did within about 10-15min). Also, it seems to really only a be a problem with our morning class (but not our afternoon class). It's very strange. I wonder how important temperature is in this process? Is it possible that if it's too hot, it simply will never solidify? Or is it possible that the plates can be too hot/cold/moist/dry? Any insight is appreciated! Thanks hive mind!

Link to this post | posted 10 Jul, 2018 21:15

c.sunnen

Hi. I'd like to ask for a bit of clarification here. I'm QCing MilleniumForce (another F1), and I've got 2 genes that both match to HTH DNA binding domains and immunity repressor when BLASTed. Both have pretty convincing HHPred data. Both are in the reverse direction. So my question is more about synteny. Which one is it? My options are (stop=34769) upstream and adjacent to Cro and antirepressor (both fwd), or further upstream than that by a few genes (stop=31573). I'm assuming it's the one directly adjacent to Cro? Thanks! Nikki

Link to this post | posted 11 Jun, 2018 14:12
c.sunnen	Okay! I'm QCing an F cluster (QuickMath) that calls a transposase. According to the approved functions list, there must be a tranposon. I don't know how to begin to check for this. Thanks!

Link to this post | posted 21 May, 2018 13:54

c.sunnen

I'm posting here because it seems logical to me to keep this MPME question within this thread. In the F1, NormanBulbieJr, we have a sequence that is only one amino acid different from the MPME1 in BPs_58. For now, I'm calling it MPME1, but perhaps it's a new one? Results below are from phagesdb: >BPs_58, function unknown, 123 Length = 123 Score = 270 bits (690), Expect = 5e-73 Identities = 122/123 (99%), Positives = 122/123 (99%) Query: 1 MFPITDTRREMTTMPTTEHESDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS 60 MFPITDTRREMTTMPTTEH SDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS Sbjct: 1 MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS 60 Query: 61 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDMLVVFDYDTSPNKAQADTADD 120 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDMLVVFDYDTSPNKAQADTADD Sbjct: 61 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDMLVVFDYDTSPNKAQADTADD 120 Query: 121 DPR 123 DPR Sbjct: 121 DPR 123