Jeff:LabNotes/Microbiome/2011-9-21
Jump to navigation
Jump to search
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