Link to this post | posted 16 Jan, 2019 18:46

c.sunnen

Okay, so I have another question regarding phage Blair. I understand to expect lysin A to be split into 2 adjacent genes, and they are (gp 16 and 17, in Blair). It's pretty clear that gp 16 is the protease domain (multiple hits in HHPred to peptidase), but it's really unclear how to call gp 17. Best matches in HHPred are to N-acetylmuramoyl-L-alanine amidase (you can view this in PECAAN), and this function is called for this pham in other phages, but no longer appears on the function list. Can I call it "lysin A, N-acetylmuramoyl-L-alanine amidase"? Or is there a better way? Thanks! Nikki

Link to this post | posted 10 Jan, 2019 14:18
c.sunnen	Thank-you both! I'll keep it as minor tail protein, then. That makes gp 12 an NKF (but is the one Debbie was suggesting could - in the future - be part of tail assembly chaperone in another thread on AN phages), and 13 is tape measure. And I also agree with Debbie's call for gene 8 being the head-to-tail adaptor (and not just a head-to-tail connector complex). Forums are awesome! Nikki

Link to this post | posted 09 Jan, 2019 16:54

c.sunnen

Hi! I am QCing Blair from Baylor University, and could use a little function guidance on gene 11 (6435-6812). There are plenty of BLAST hits to minor tail protein, and there are some HHPred hits that support "tail component" (see attached screenshot), but there's an equivalent HHPred hit to minor capsid. It's also too small for standard minor tail proteins (351bp)and is upstream of tapemeasure (not where I'd expect; there is a minor tail protein downstream of tapemeasure that fits all the criteria, but only one). Keep in mind, this is a small genome, with only 26 genes! So is this really a minor tail protein? Some other tail component but we can't be sure so it's NKF? Could it be a minor capsid protein? Thanks for the insight! Nikki 28Kb

Link to this post | posted 23 Oct, 2018 21:03

c.sunnen

Just thought I'd share that we got great DNA concentrations and quality today with our first attempt with this protocol. We only used 2mL (not the 5 our protocol says), and got concentrations around 250ng/ul, with A260/280 of 2-ish. Very pleased! Thanks for all your help! Now to run it on a gel with/without RE buffers… Nikki (I've attached our protocol here, in case anyone else wants to try it). 16Kb

Link to this post | posted 13 Oct, 2018 00:01

c.sunnen

Sorry to keep bugging, but I'm revising our protocol: what's the concentration of Proteinase K? The instructor guide protocol calls for 5uL per 1mL sample, but it doesn't have a concentration. Also, you recommended above 0.5M EDTA, but the protocol says 0.1M. Is one concentration preferred. Thank-you again for all of your input, and thanks Maria for your insight! If we have trouble, we'll probably eliminate the nuclease step, too! Nikki

Link to this post | posted 12 Oct, 2018 19:18
c.sunnen	Thank-you, thank-you, thank-you!!!

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