Link to this post | posted 16 Nov, 2016 16:21
viknesh	Dan, here is a representative gel (of ~ 40 gels) from the workshop when faculty used the new DNA isolation protocol. As you can see, there is some amount of smear. Is this what you are receiving for sequencing? 140Kb

Link to this post | posted 11 Nov, 2016 15:08
viknesh	Hi Laurie, Is this true across the board, or are only 3 students getting low DNA yields? A little more cumbersome, but you could try to concentrate the phage, either using the EM protocol for concentrating phage, or the PEG precipitation protocol. When concentrating, you should spin at 4C. Vic

Link to this post | posted 09 Nov, 2016 21:24

viknesh

Rodney King Has anyone experienced degradation of DNA isolated by the new protocol? We tried a brand new DNA isolation kit and brand new enzymes and still see degradation. Never experienced this problem before. The fact that the DNA disappears only in the presence of restriction enzyme buffer indicates DNase is not being thoroughly removed. Hi Rodney, I had a conversation with Steve Caruso today, and he's seen what you've been describing. He thinks it happens when students remove the resin/denaturant without first properly mixing the solution. As a result, the DNase added to the lysate/resin mix may not be fully denatured. Any remaining folded DNase may be inactive during purification because of the presence of EDTA. In the elution (in water), perhaps there isn't sufficient cations to support DNase activity. The addition of the restriction buffer, however, bring in cations that might support DNase activity. Thoughts? Vic Edited 10 Nov, 2016 00:28

Link to this post | posted 08 Nov, 2016 18:38

viknesh

Hi Cathy, Are you getting low yields across the board or just for a number of students/samples? Typically, one can expect a yield of ~ 40ng/ul or higher from a 10^9 pfu/ml lysate when prepping using the protocol 9.1. If a lysate yields low DNA, there is always the option to concentrate the lysate by centrifugation (see PEG precipitation, or the TEM protocol). Regardless of the protocol you use for concentrating phage by centrifugation, it will be important that you spin at 4C. If you are seeing low yields across the board, perhaps its because of a bad reagent. Vic

Link to this post | posted 03 Nov, 2016 18:35

viknesh

Hi Rodney, I can't say I've seen this, and have not yet heard from anyone with this problem. Perhaps someone who has will chime in. In the meanwhile, would you share a gel where you see the degradation only in the presence of restriction enzyme buffer? I assume the buffer isn't contaminated with DNAse, and that you are using brand new buffer when you used the new enzymes? Vic Edited 03 Nov, 2016 18:36

Link to this post | posted 02 Nov, 2016 15:51
viknesh	Emily, do you have a lysate of arthrobacter phage that you use as a control? Has this also stopped producing plaques? Vic

Link to this post | posted 02 Nov, 2016 15:07

viknesh

Hi Emily, We know that Arthrobacter lose viability when stored on plates or in liquid culture over time. In the A. sp Phage Discovery Guide, I think the guidelines are to use colonies or cultures that are no older than 2 weeks in order to obtain consistent results. The phage we have isolated on Arthrobacter, however, have been pretty stable in lysates. It is surprising that your students are not able to recover any phage from 2-3 week old plates. Is it worth flooding the plate 3 mls media, collecting 1 - 2 mls of the lysate, and the trying to infect with that? Are you able to streak purify directly from a plaque? Vic Edited 02 Nov, 2016 15:07

Link to this post | posted 27 Sep, 2016 18:42
viknesh	Christine, While you are doing your tests, it may be worth asking for smeg from your buddy school. That way, you'll at the very least have a strain to compare to. Vic

Link to this post | posted 22 Sep, 2016 21:09

viknesh

Hi Christine, It's going to be hard to tell what you have without photos of your plates with colonies. I've attached a figure with smeg on streak plates over time when incubated at 37C. You'll see from the figure that small specks become visible at day 2, and they gradually grow into larger well-defined colonies with a dry and irregular morphology by day 4 - 5. They are white to tan, depending on the age of the colonies and the properties of the plate. An important test for you to perform is a plaque assay (or a spot test) with D29. If you see plaques or a cleared spot on your bacterial lawn, then you can be confident that you are working with smeg. Vic 686Kb

Link to this post | posted 07 Jul, 2016 21:26

viknesh

Hi Emily, I've seen this happen before - lawns and cultures of Arthrobacter take an extra day or two to become dense or saturate, and lawns tend to be thin and spotty/clumpy. We found that this happened unexpectedly from time to time when we grew cells at 30C. Once we switched to doing everything at 26C, this problem went away. I suggest starting from your glycerol stock and try to do everything (streak, culture in liquid, plate and incubate) at 26C (or at room temperature). I'll be curious to know if this solves the problem for you too. Vic