My bacteria keep dying and it's making me sick!

Re: My bacteria keep dying and it's making me sick!

• LauraT25

  

  February 2010

  Have you checked to make sure your cells are still competent?  Sometimes they go bad, you may need to make more (or buy more).  You can do a test with a control vector to make sure it's not your plasmid.  Did you get the plasmid from another lab?  If so, you may want to ask them what cells they use, their concentration of antibiotic, and the temperature they're growing - some weirder plasmids work best in more unusual competent cells like RILs, or lower temp or lower antibiotic concentration.  It's defintiely a crapshoot with a new plasmid.

  
  

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• LauraT25

  

  February 2010

  By your post, I'm assuming you're not inducing them to make protein, you're just trying to get out the DNA?  If you're inducing, there's a whole new host of issues since sometimes the protein produced is lethal.  Sorry, I know you came here to vent, and I DO empathize, it totally sucks.  I've been doing this for four years now, I feel your pain.

  
  

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• nefariousmango

  

  February 2010

  Wow, an informed reply!  Luckily no one's transformations are working lately, including those done by the post-docs who've done them for years, so we're missing something bigger or our oligos are just especially finicky.  We know that these ligations are not effcient so the best we've ever gotten is about 15 colonies, which is not far from 0 so... yeah.
  
  Yes, the cells are competent (made them last month and have checked them numerous times) and I made the vector and have checked it almost weekly; when I've transformed cells with it uncut they live.   If I digest it and then do a ligation the cells die and when I run out the digest on a gel I get two clear bands where I'd expect them to be, so I'm pretty sure my restriction enzymes are both working and my vector is pure.  I'm digesting/ gel purifying more this week and will run those controls again, just in case.  We also ordered fresh T4 ligase and that seems to help- I got a whooping 2 colonies this week, a post-doc got 1, so better than none!
  
  This week: miniprep, digest, PCR my 2 colonies to see if they're even what I want, transform in a richer LB medium (add glucose) and see if that helps, and use a higher DNA:cell ratio in my transformations.
  
  Oh, and btw, I'm doing shRNAs for a txn factor in the p53 pathway, so I'm not trying to get protein expression.
  
  What kind of research do you do?
  

  

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• LauraT25

  

  February 2010

  Jeez, it sounds tricky.  Sounds like the ligation and REs are working, so I can't begin to guess what's wrong.  Bummer, it would have been awesome if a stranger on the internet solved your problem, right?
  
  Funny, my lab actually works on p53 too, but somewhat peripherally - I guess that's not too unusual though, considering.  My lab studies a family of calcium-binding proteins (they work similar to calmodulin) and one of them is involved in cancer and happend to bind p53.  That's the one I work with, though I work on structure/function type research, so I study the binding of my protein to various compounds, in theory inhibitors that will suppress the p53 interaction and 'cure cancer'.  Lots of binding studies, plus structural studies (NMR, X-ray).  I haven't had to optimize a vector in a while, I only use bacteria anymore to make new protein, but in the past I've done a lot of subcloning and DNA and RNA purification, so I feel your pain.  No shRNA so far, generally the 'cell culture girl' in my lab does most of that kind of thing.
  
  Sorry, that got kinda long.  That's what happens when you get two dorks talking about science

  
  

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• aggiebug member

  

  February 2010

  ahhh so frustrating!! I have never done the research so I can't give ya any help.  But I get what you are doing and why that is frustrating so I can sympathize  

  Love is like infinity: You can't have more or less infinity, and you can't compare two things to see if they're "equally infinite." Infinity just is, and that's the way I think love is, too.
  Fred Rogers

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• nefariousmango

  

  February 2010 edited February 2010

  Laura: Yeah, I guess RAS and p53 are the major players when it comes to cancer and "curing" cancer is a common goal, so it's not surprising.  We're really trying to pinpoint why nutlins don't work in reality the way they do in theory, at least most of the time.  So we're semi-systematically inhibiting molecules to figure out what's different between traditional chemo-treated cells and nutlin-treated ones.
  I don't know anything about protein research techniques, I mean I've learned about them but it mystifies me how you can study folding and structure and such! 
  
  aggiebug- thanks for the sympathy!  I need it sometimes!

  

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• zaneopal member

  

  February 2010 edited February 2010

  We were having the same problem last semester, too. (We work with bacterial quorum sensing) For us, it did turn out that our cells were no longer competent, though maybe your plasmid is cutting a vital gene? I can't remember if someone suggested this already, but go back and what REs you're using. It's possible it's cutting the DNA in more than one place and not religating....granted if you're using E. coli that's probably unlikely, but it's all I can think of right now.
  
  Edit to add another thought: How are you transforming them? Just heat shock? Because the grad student who is supervising me was having your problems too. We electroporated last week and got a significantly higher yield, so maybe try that too?

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• nefariousmango

  

  February 2010

  We're actually trying to replicate plasmids we've constructed so they are not actual e/ coli genomes, just vectors w/ amp resistance cassettes and a little peice of RNA designed to block a gene in human cells.  So, we want two cuts!  As for the competence, see my abpve reply about our controls thus far.
  
  I think electroporation is usually more efficient, but we don't use it in our lab at all and until recently there haven't been any problems like this using just heat shock!
  

  

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• LauraT25

  

  February 2010

  Yeah, if you don't already have the setup for electroporation that won't be so simple, but I have heard it works a little better.  I know there are protocols for chemical transformation as well, but I've never tried it myself, we always just stick with heat shock.  Sometimes 1-2 colonies is just what you're going to get, though it kind of sucks!
  
  I used to work in a lab that used mass spec to study the secondary structure of RNA hairpins (long UTRs, not shRNA) and I commend you, working with RNA can be a real PAIN.  We inserted DNA and used T7 to make the RNA, so it was a bit different, but RNA is still super finicky and I'm glad I don't have to be so super-clean anymore!
  

  
  

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