Welcome to the forums at seaphages.org. Please feel free to ask any questions related to the SEA-PHAGES program. Any logged-in user may post new topics and reply to existing topics. If you'd like to see a new forum created, please contact us using our form or email us at info@seaphages.org.
Recent Activity
All posts created by viknesh
Link to this post | posted 22 Oct, 2018 13:13 | |
---|---|
|
smolloy123 That's a really neat trick, Sally. Do you know if it works for M. smeg too? I'll definitely give it a try and report back. |
Posted in: Gordonia → Clumping in liquid cultures
Link to this post | posted 14 Oct, 2018 16:34 | |
---|---|
|
c.sunnen I edited my post above to reflect the correct concentration (should someone else refer to it). |
Link to this post | posted 13 Oct, 2018 02:47 | |
---|---|
|
c.sunnen Thanks for pointing that out. The concentrations were only included in the Discovery Guide, and not in the Instructors Guide. It's now been updated. |
Link to this post | posted 12 Oct, 2018 17:50 | |
---|---|
|
c.sunnen Ah, I now see that you intend to complete the protocol by precipitating DNA instead of using the Wizard columns. You are correct - you can go ahead and resuspend in TES, then add ProtK. In the zinc chloride precipitation protocol, we use EDTA instead of TES just to make things easier for those wanting to precipitate with zinc and continue with the old protocol (that is, they do not have to make TES). However, considering that you will use both zinc and protK, and therefore EDTA and SDS, using TES makes sense. The conc. of SDS recommended for ProtK is 0.5% w/v. Perhaps you could prepare your TES to contain 0.1M EDTA and 0.5% SDS. |
Link to this post | posted 12 Oct, 2018 15:22 | |
---|---|
|
c.sunnen Hi Nikki, A couple of things. For M. foliorum, it is important to use ProtK and SDS to remove residual nuclease activity from DNA preps. Residual nuclease is not a problem during the prep process itself, since EDTA is present. Once eluted in water, the lack of EDTA is still not a problem since the nucleases have no access to metal cofactors required for activity. However, any downstream analysis using buffers will activate the nucleases. If you can get away with the standard DNA extraction protocol (wihtout a precipitation step), I would encourage that because we've tested that protocol (with ProtK and SDS) extensively over the summer. We have some, but significantly less, experience using ProtK with the zinc precipitation protocol. If you do decide to continue with the zinc chloride precipitation protocol, here are some things to note. With the zinc chloride protocol, EDTA has to be added after the precipitation step, since EDTA will chelate the zinc that is needed for the precipitation step. Currently, the Instructors Guide suggests the following: Add nuclease to the lysate -> precipitate with zinc chloride -> resuspend in EDTA -> add ProtK + SDS to the lysate -> continue with standard prep. One issue with this workflow is that the precipitation step results in a significant fraction of particles releasing DNA during the resuspension step. It is therefore critical that EDTA be used for resuspension, and that you work quickly to add the EDTA. Otherwise, any residual nuclease will rapidly degrade DNA that is released during pellet resuspension. In the Instructors Guide, I recommed using the "alternative" strategy for Step D1. Once resuspended in EDTA, you can then add ProtK and SDS to remove residual nuclease. Another workflow would be as follows: nuclease-treat the lysate -> add ProtK + SDS to the lysate -> precipitate with ZnCl -> resuspend in EDTA -> continue with standard prep. I've not tested this workflow. The hope here is that ProtK will remove all nuclease activity prior to precipitation, allowing you to work more slowly during the resuspension step. However, my fear here is that SDS might destabilize some fraction of phages, in a phage-dependent manner, and you'll end up losing a significant fraction of DNA to the supernatant during the precipitation step. If you have a student that has extra time and wants to help test this, let me know Vic |
Link to this post | posted 10 Sep, 2018 15:39 | |
---|---|
|
Hi Jake - thanks for writing. The app is down at the moment, and will likely not be back up again for a while. We suggest that you have your students record the information in their lab notebooks, and use their smartphones or computers to determine the GPS location of their collection sites. We've removed instructions for using the app from the Phage Discovery Guide. |
Posted in: SEA-PHAGES App → a work in progress
Link to this post | posted 08 Sep, 2018 02:16 | |
---|---|
|
Hi Evan, We've posted a modification that we know works for extracting DNA from M. foliorum phages. The modification is listed as an optional step in the DNA extraction protocol, and involves adding ProteinaseK and SDS. Vic |
Link to this post | posted 27 Aug, 2018 18:13 | |
---|---|
|
kaylafast I would be suspicious of this streak plate (and the culture from which the streak was setup). There appears to be more growth than expected. Go ahead and test your latest culture, but also setup streak plates (and mock streaks) from your glycerol stocks. Let me know what you see. You can then go ahead and setup cultures. |
Link to this post | posted 27 Aug, 2018 15:59 | |
---|---|
|
kaylafast Kayla, can you include the incubation duration before the pictures were taken, for each plate? |
Link to this post | posted 27 Aug, 2018 15:43 | |
---|---|
|
kaylafast Hi Kayla, A few things: 1 - Would you be able to share pictures of your streak plates (both from glycerol but also from liquid culture)? You'd expect them to behave similarly, with growth perhaps faster for the plate streaked from the liquid culture. 2 - From the pic you attached, it is hard to tell if a) there is a robust bacterial lawn but turbid plaques, b)light/non-robust lawn with turbid plaques, or c)light/non-robust lawn with clear plaques. If the plaques are turbid, it is likely that you have a contaminating microbe in your liquid culture that is not lysed by D29. When you setup liquid cultures, it maybe worth setting up a mock culture. For this, you do exactly what you do as you are setting up your liquid culture, but instead of picking up a colony with an inoculating stick/loop and dipping in into the liquid media, you just dip a sterile inoculating stick/loop directly into the media. This way you can determine if your flask, media, or inoculating loop is contaminated. Vic |