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
JustinA posted in Plaques appear on spot test, don't appear in titer
Viknesh Sivanathan posted in Plaques appear on spot test, don't appear in titer
JustinA posted in Plaques appear on spot test, don't appear in titer
Viknesh Sivanathan posted in Plaques appear on spot test, don't appear in titer
RyanWheeler posted in Plaques appear on spot test, don't appear in titer
All posts created by debbie
Link to this post | posted 06 Nov, 2015 18:49 | |
---|---|
|
Hi Joe, I heard that you spoke with Kevin yesterday about this. I am just adding a link to the protocol that is already on seaphages.org. http://seaphages.org/media/docs/NewDNAProtocol.pdf |
Link to this post | posted 15 Sep, 2015 17:41 | |
---|---|
|
so it sounds like you are testing your post-enrichment plates by putting 50 ul of sample into smeg and plating. Are you also doing spot testing? Sometimes 50 ul of enriched samples can so 'blow-out' the cells on a plate, you see no plaques. If the plaques are turbid, you will see lighter confluent growth of cells, so it can be quite confusing. Just thinking. |
Link to this post | posted 15 Sep, 2015 12:24 | |
---|---|
|
Also - what flasks are you using for growing smeg? |
Link to this post | posted 15 Sep, 2015 12:23 | |
---|---|
|
Greg, Hi. In your final working cultures and media, no Tween is recommended. (nor needed, unless you are having issues. I have never added Tween to anything but my liquid cultures.) So no Tween in the Top agar, Luria agar or broths used to grow the cultures. Tween is used when you are making the 'starter' cultures for your big plating batches of smeg. By that I mean, I inoculate a series of 3-5 ml cultures tubes that contain media (L or 7H9, antibiotics, ADC, and Tween -Calcium if you want)from single colonies on my streak plate of smeg. That gets the smeg into liquid with no clumps. I let those grow up until they are a thick culture. I discard any of these that are clumpy. Wwith the best heavy growth liquid smeg, I sub a small aliquot (usually a drop) into the larger volume of media WITHOUT TWEEN that I will then use for plating. Make sense? |
Link to this post | posted 15 Sep, 2015 11:36 | |
---|---|
|
Hi Greg, So without seeing, I can only guess. If you have no plaques with D29, it could be that you are not growing smeg. Streak out the liquid culture. If there are ANY colonies in 24 hrs., it is not smeg - or the smeg is contaminated. So to be thorough, culture everything to be sure nothing is contaminated. Start your smeg cultures from a single colony and follow regular protocols. |
Link to this post | posted 06 Sep, 2015 01:06 | |
---|---|
|
Hi Greg. Tween can also be used by the bacteria as a carbon source, so it will be broken down by the time you plate for phage. I would not fret too much about using a Tween culture for enrichment. Sharon is correct that Tween can inhibit phage adsorption, but once it is hydrolyzed, there should be no worries. debbie Hatfull Lab |