Daniel:Notebook/HiResChrPaint/2014-3-5
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Overload FISH[edit]
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]
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]
- Prepare a cell suspension of 500 000-2 000 000 cells/mL in RPMI
- Add 20 uL of cell suspension per dish to the cener; add 80 uL RPMI
- Allow cells to adhere for 6 hours at 37 C
- Rinse slides briefly in 1X PBS
- Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
- Rinse briefly in 1X PBS
- Incubate for 5 minutes in 1 mL of 2X SSCT
- Incubate for 5 minutes in 1 mL of 2X SSCT + 50% (v/v) formamide
- Remove media and add 1 mL of 2X SSCT + 50% (v/v) formamide for storage at 4C; wrap with parafilm
- Slides are typically best used within 1-2 weeks of creation