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  2. → Viewing Profile: Posts: cspD

cspD

Member Since 30 Nov 2012
Offline Last Active Dec 08 2012 05:59 PM
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Posts I've Made

In Topic: Questions about overnight cultures

01 December 2012 - 08:03 PM

View PostJohn Forsberg, on 01 December 2012 - 06:31 PM, said:

View PostcspD, on 01 December 2012 - 05:48 PM, said:

No my mutants have a transposon inserted into the genome in the particular gene. So I wont run into this problem.

How do you select for your cells over contaminants?  I'm not familiar with the techniques you're referring to.  It looks like I'm out of my depth here, so I can't really say whether going directly from a glycerol stock would be okay with your system.

When I first made my stocks I streaked my mutants on plates w/ gentamicin, then inoculated into broth which I froze down. I don't use antibiotics in the broth when I am sub culturing for an experiment. Contamination never really has been an issue, besides for the wild type, there is no resistance marker anyway.

In Topic: Questions about overnight cultures

01 December 2012 - 05:48 PM

View PostJohn Forsberg, on 30 November 2012 - 05:34 PM, said:

Do you not have plasmids with antibiotic resistance markers in your bacteria?  The main reason I've heard for keeping your cells from growing too long is to avoid cells without your plasmid from overtaking your cultures (as the antibiotics will be progressively degraded over time).  I suppose even bacteria without plasmids will get further into stationary phase (with many more cells that are dead) the longer you incubate them as well.

No my mutants have a transposon inserted into the genome in the particular gene. So I wont run into this problem.

In Topic: Questions about overnight cultures

30 November 2012 - 03:05 PM

View Postbob1, on 30 November 2012 - 01:57 PM, said:

The way you have been doing it is the correct one, however the reasoning behind doing the culture straight from the glycerol stock is that it was made from a single clone in the first place, therefore it shouldn't need to be re-streaked.

For plasmids the optimium time is about 16 hours of overnight culture.

Yea that's what I was thinking. My freezer stocks were frozen down from liquid cultures from a single colony. So theoretically they should be from the same clone. So is it safe to just inoculate broth cultures from frozen stock? Also if I'm not doing plasmid work, is it alright to leave for 18 hrs? I ask because some people are really strict and say 16 hrs is the maximum

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