Janet B. Matsen:Open Lab Questions

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do? (7/13/2012))
(What causes an overloaded colony PCR reaction to fail?)
Line 13: Line 13:
* It is simple to do an experiment where you transform some without waiting and some after waiting, of course.
* It is simple to do an experiment where you transform some without waiting and some after waiting, of course.
-
===What causes an overloaded colony PCR reaction to fail? ===
+
 
===How does oxygen limitation cause lower plasmid copy number? What about the growth medium?  ===
===How does oxygen limitation cause lower plasmid copy number? What about the growth medium?  ===

Revision as of 21:45, 17 November 2012

Return to Janet

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!

Contents

Open Questions

Why does the DNA clump when you run overloaded agarose gels? (2012/10/02)

polka-dot DNA in a 0.7% agarose gel
polka-dot DNA in a 0.7% agarose gel

Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do? (7/13/2012)

  • 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.."
  • online: ""After making the cells leaky, the DNA is added to the cells and allowed to sit on ice for 10-20 minutes. This allows the DNA to get past the cell membrane, and gives enough time for lots of cells to recieve the DNA and to make certain all of the DNA gets in the cells. After this, in order to make the cells keep the DNA, and to make certain they survive, (Being leaky is not a good thing) the cells are heat shocked for several seconds to 'turn on' (induce) heat shock genes which aid in survival and recovery. After that, the cells are incubated to start growing and plated on selective media to recover those cells that actually recieved the DNA.
  • It is simple to do an experiment where you transform some without waiting and some after waiting, of course.


How does oxygen limitation cause lower plasmid copy number? What about the growth medium?

  • TB gives way higher yield than LB
  • Growing cultures with liquid:air volumes higher than 1:5 is very bad for plasmid yield.

Why do apples make people burp?

  • (posted 5/23/2012)
    • I have been wondering this for years.
Personal tools