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5:35 pm
August 24, 2010
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Dan Russell
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| posts 44 | |
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Hi all,
Welkin asked me to post a couple of protocols to get things started.
Here's a protocol on how to get usable PCR template from a smeg culture.
Here's another one which carries the details of how we do PCR on smeg (or lysogen!) template.
Keep in mind that if you use the first protocol to prep template, you'll use 10 microliters for PCR (not the 1 specified in the PCR protocol itself).
–Dan
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5:40 pm
August 26, 2010
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Debbie
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| posts 12 | |
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Hi all,
Here are two protocols for creating lysogens. There is no magic to this, just lots of thoughtful considerations and verfications along the way to ensure you have a real lysogen.
Lysogeny Assay: This protocol will yield an efficiency of lysogeny. If you use it to create lysogens, it may prove problematic if your lysate titer is not sufficient. As beginners, we recommend the next protocol to create your lysogens.
Creating and Verifying Lysogens: This protocol is our preferred method for creatiing lysogens because you will actually see bacteria in the center of the spot.
Good luck! debbie
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4:51 pm
September 2, 2010
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Welkin
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| posts 9 | |
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Hello All,
Here is the immunity assay protocol that Kurt asked for.
Let me know if there are any more questions!
Welkin
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11:32 am
November 11, 2010
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rdejong
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| posts 4 | |
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For the creating and verifying lysogen protocol, we have a couple questions:
1) should we be using plates with 7H9 instead of LB as the bottom agar?
2) the liquid culture for culturing mesa-derived lysogens, is that 7H9 complete? How much Tween?
3) Does "killing from without" refer to lysogens that have gone lytic?
Thanks,
Randy
(Calvin)
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4:48 pm
November 11, 2010
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Debbie
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| posts 12 | |
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1) should we be using plates with 7H9 instead of LB as the bottom agar?
I believe you should be able to proceed just fine using LB since you initially isolated on LB. If it doesn’t work, let us know. Welkin
I don’t think it matters. But that will prove true as the experiment continues (i.e. We here at pitt (using 7H9 get different results – that is if we do the experiment here), I wouldn’t go out of my way about this, but the proof will be in the pudding!… debbie
2) the liquid culture for culturing mesa-derived lysogens, is that 7H9 complete? How much Tween?
Yes, complete 7H9. We add 250ul of 20% Tween 80 to 100ml of medium. welkin
we only use Tween to make sure the bacteria doesn’t clump when it goes into culture. You can grow your bacteria in LB too. debbie
3) Does "killing from without" refer to lysogens that have gone lytic?
No. “Killing from without” refers to the clearing you sometimes see when you spot a very large amount of phage on a lawn. If the titer of the phage spot is high enough, you may kill the cells without actually having a productive infection— kind of like just dropping a big bunch of needles on balloons. Even though a single phage might not be able to infect that type of host cell, a whole ton of them can overwhelm the cells and kill them anyway— without the formation of any new phage. You can tell the different through serial dilutions— a high titer stock that is “killing from without” will not produce single plaques at lower dilutions. welkin
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5:53 pm
November 11, 2010
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lhughes
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| posts 10 | |
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I told this to Randy in e-mail, but just for everyone else – we isolated our Adephagia lysogen on LB.
Lee
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