Daniel:Notebook/HiResChrPaint/2014-3-5

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Overload FISH[edit]

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From the results last week, I believe that the probe sets may need an increased signal in order to be visualized. In that spirit, I'm going to amplify more probe and use a lot of it on one experiment.

qPCR[edit]

  10 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V61U 100 uM AP2V6 2 mM aa-dUTP H20 Total Volume
per rxn 0.4 50 0.4 0.4 2.5 46.3 100
V6S2 (6.1x) 2.44 305 2.44 2.44 15.25 282.43 610
V6S3 (6.1x) 2.44 305 2.44 2.44 15.25 282.43 610

qPCR Results[edit]

File:2014-03-05-V6S2V6S3.png

Nanodrop Results[edit]

Sample V6S2A V6S2B V6S3A V6S3B
ng/uL DNA 78.9 77.5 85.2 86.2
ug in 40 uL 3.2 3.1 3.4 3.4
ug total   6.3   6.9


Slide Preparation[edit]

  1. Prepare a cell suspension of 500 000-2 000 000 cells/mL in RPMI
  2. Add 20 uL of cell suspension per dish to the cener; add 80 uL RPMI
  3. Allow cells to adhere for 6 hours at 37 C
  4. Rinse slides briefly in 1X PBS
  5. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
  6. Rinse briefly in 1X PBS
  7. Incubate for 5 minutes in 1 mL of 2X SSCT
  8. Incubate for 5 minutes in 1 mL of 2X SSCT + 50% (v/v) formamide
  9. Remove media and add 1 mL of 2X SSCT + 50% (v/v) formamide for storage at 4C; wrap with parafilm
  10. Slides are typically best used within 1-2 weeks of creation