Jeff:LabNotes/Microbiome/2011-9-21

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Single Mammalian Cell Amplification: Ideas[edit]

  • I want to apply my device to mammalian cells as well
  • It would be cool if we could use it for single stem cells or cancer cells

Contamination

  • Since currently I have ~ 50% human contamination rate, I cannot trust the results from human cells
  • I will use mouse embryonic fibroblasts (MEFs) from the Xu lab
  • Alan will help me grow the first batch and hopefully I can take over maintaining this cell line

Visualization

  • These cells are much bigger than bacterial cells
  • I can spread them, stain with SYBR green, and visualize to get a good idea of the proper concentration to use.

Process

  • Everything can be done using the exact same method as with bacterial cells
  • The cell lysis will be different since there is no cell wall in mammalian cells. Freeze/thaw lysis will not be used
  • It would be advantageous to use a strong base/alkaline solution for cell lysis as this is needed for denaturing and will remove the need for adding additional reagents.
  • Based on previous papers, I can use something similar to an ALS buffer: 200 mM KOH, 50 mM DTT, and incubate at 65 C for 10 min
  • ALS mix: 4 uL 5M KOH, 5 uL 1M DTT, 91 uL water
  • Then use the following NS buffer: 900 mM Tris-HCl, 300 mM KCl, 800 mM HCl
  • NS mix: 4.5 uL 2 M Tris-HCl, 750 uL 4 M KCl, 66.67 uL 12N HCl, 4.683 mL water
  • The MDA, library construction process should be the same.

Applications

  • Look at CNV's
  • Look at cell diversity at different embryonic stages