Janet B. Matsen:Open Lab Questions

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(Open Questions)
(Open Questions)
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*Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA?  What does this do?  
*Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA?  What does this do?  
**[http://partsregistry.org/Help:Transformation_Protocol| Example protocol] showing ice incubation
**[http://partsregistry.org/Help:Transformation_Protocol| Example protocol] showing ice incubation
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** Mila weighing in: "This waiting period is part of the protocol. Not sure anyone knows what exactly happens but likely DNA just needs this time to land onto a cell. Longer time does not do much for the efficiency of the transformation. However the efficiency of the cells is determined by something upstream, by how the cells were grown and chemically treated. This is the most important part of the transformation process.."
*What causes an overloaded colony PCR reaction to fail?   
*What causes an overloaded colony PCR reaction to fail?   

Revision as of 15:08, 14 July 2012

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These questions are "open" to me, not necessarily the scientific community. If you have insights or can point me toward references relevant to them, please do!

Open Questions

  • Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do?
    • Example protocol showing ice incubation
    • Mila weighing in: "This waiting period is part of the protocol. Not sure anyone knows what exactly happens but likely DNA just needs this time to land onto a cell. Longer time does not do much for the efficiency of the transformation. However the efficiency of the cells is determined by something upstream, by how the cells were grown and chemically treated. This is the most important part of the transformation process.."
  • What causes an overloaded colony PCR reaction to fail?
  • How does plasmid copy number get affected by oxygen availability? Cellular media?
    • I have noticed that LB often gives me orders of magnitude lower plasmid yields and that growing cultures with liquid:air volumes higher than 1:5 is not good.
  • How does colony density affect colony size?
    colony size varies with colony density
    colony size varies with colony density
    • I observe that when there is a large number of colonies per area on an agar plate, that the colonies are smaller. I have thought about the possibility of diffusion-limited arrival of nutrients, however, one would expect to see larger colonies on the outside of the cluster if this is the case.
  • Why do apples make people burp? (posted 5/23/2012)
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