EricChu:LabNotesMDA/2013-3-25

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Using MEF and High Concentration Phi29 in the Vertical Chamber Device[edit]

  • pH adjustment for device was tested prior to this experiment. pH of ALS+NS should be = 6.0 to get a neutral pH in the device.
  • 2 cells were captured in the capture chamber. Cells were lysed in the device.
  • Air pushing lysed sample to the next area. But visible amount of samples remained in the mixing rings.
  • Mixing MDA mix and cell contents on device. No observable DNA.
  • After 5 hours 30C on TE module, clusters of DNA were visible in the high vertical chambers but not the rounded channels. However, the number of clusters (visible DNA) were lower in this experiment.
  • 4-5ul of samples were collected from each chamber.


File:03252013qPCR.jpg


  • Images of DNA clusters in different chambers
    • DNA contents of 2 MEF cells were distributed in 24 chambers
Chamber 1 Chamber 2 Chamber 3
File:03252013ch1.jpg File:03252013ch2.jpg File:03252013ch3.jpg


Result[edit]

  • In both experiments on Mar20 and Mar25, visible DNA clusters were seen in all vertical chambers. However, there were almost none visible in the rounded chamber areas.
  • All chambers had similar amount of DNA clusters. But the amount of mouse specific DNA was low.
  • Two possible reasons:
    • 1. DNA amplified was not of mouse original. The positive control human Alu primers did show the presence of human DNA. (It can be tested with real time MDA to quantify the amount of total DNA)
    • 2. The total amount of amplicons was low. (It may due to the final condition, such as pH, in the chamber.)


  • I'll test the same samples with another set of mouse specific primers.
  • I'll run another test with normal Phi29 concentration.
  • I'll design the device with larger final MDA chambers for more reagents. (I wonder if I could just increase the amount of dNTP to boost up the final amount of product)