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Cloning issue with bacteria growing in primary media but not in secondary


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#1 Kyle Strickland

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Posted 13 February 2016 - 06:56 AM

Okay so I have been working on a triple ligation for a little while and we are opting for the ligation and transformation method. We are using electorporation as the means to get the vector into the top-10 cells. The resistance for selection is Kan and one would think that if the bacteria grows on the plate, well it should grow in the liquid media for mini prep. Not in my case, I have about 1 our of 10 colonies growing and its not like there are satellite colonies or anything. Only think I can think of is the cells are making a biofilm for protection or the gene they have uptaken has a weak resistance for kan. I have made the plates multiple times and we use the same ratio for solid media and liquid media 1:1000 from our stock. I am about to abandon this method in favor of the PCR amplification method. Any ideas on why colonies would grow on LB Kan30 agar but not in LB Kan30 liquid media?



#2 labtastic

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Posted 15 February 2016 - 06:28 AM

Is it possible the insert you are ligating is toxic to the cells? 

 

Other than that, my best guess is that your ligation isn't working (or is working very efficiently...not surprising with triple ligations) and the few colonies you are getting on your plate just simply don't have your plasmid and have somehow acquired low resistance. I've seen this on rare occasions, but don't have a good explanation for it.

 

When you say triple ligation, I assume you are ligating plasmid backbone to two fragments? I would fuse the two fragments by overlap extension pcr first and then do the ligation with the plasmid and single insert. It'll be much more efficient.


Edited by labtastic, 15 February 2016 - 06:28 AM.


#3 miST32

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Posted 17 February 2016 - 09:38 AM

I'd try a few things:

1.  Lower the temperature to 32C.  The insert product may be toxic, but so too may the DNA cause problems with replication.  Sometimes lower temperatures can help both problems.  If DNA instability is a potential cause, consider amplifying the plasmid in cells such as NEB Stable or Stbl3.

2. Try the liquid culture in different vessels (different plastic, pyrex, etc).  Sometimes (particularly with reusable tubes or flasks) contaminants might sneak in.

3.  Streak out a colony onto a fresh plate with higher [Kan].  It should grow well at 50ug/mL if it is Kan-resistant.  This could help tease out false resistance or low-resistance.  Streak out your competent cells alone (without transforming them with a Kanplasmid).  Use a different source of competent cells if you get growth on both plates.

Good luck!



#4 Andrea Fortina

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Posted 08 June 2016 - 01:03 AM

Do you try to ligate the fragment one by one, not just in one step?


Andrea Fortina

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