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.
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10 nM OligoPool
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2X Kapa SYBR qPCR MM
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100 uM AP1V61U
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100 uM AP2V6
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2 mM aa-dUTP
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H20
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Total Volume
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per rxn
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0.4
|
50
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0.4
|
0.4
|
2.5
|
46.3
|
100
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V6S2 (6.1x)
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2.44
|
305
|
2.44
|
2.44
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15.25
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282.43
|
610
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V6S3 (6.1x)
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2.44
|
305
|
2.44
|
2.44
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15.25
|
282.43
|
610
|
qPCR Results[edit]
File:2014-03-05-V6S2V6S3.png
Nanodrop Results[edit]
Sample
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V6S2A
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V6S2B
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V6S3A
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V6S3B
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ng/uL DNA
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78.9
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77.5
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85.2
|
86.2
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ug in 40 uL
|
3.2
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3.1
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3.4
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3.4
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ug total
|
|
6.3
|
|
6.9
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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