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Dye Labeling[edit]
Time for dye labeling of the padlock probes. I'm going to label the padlock probes with Alexa 488 and I'm going to make a Cot1-546 for controls.
Sample Matrix
Sample
|
uL Sample
|
ug Sample
|
uL ULS 488
|
uL ULS 546
|
Cot1
|
2
|
2
|
0
|
5
|
PPRL
|
40
|
1.4
|
2
|
0
|
- Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C)
- For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C)
- VORTEX THESE SOLUTIONS VIGOROUSLY BEFORE USE EVERY TIME!
- Resuspend DNA in 20 uL labeling buffer (Component C)
- Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
- Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
- Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
- Purify using a column (Centri-Sep recommended)
File:ULYSIS ReactionTable.png
Labeling Results[edit]
Sample
|
ng/uL ssDNA
|
pmol/uL dye
|
pmol/uL DNA
|
dye/probe
|
bp:dye
|
PPRL-488
|
33.5
|
0.6
|
1.0
|
0.6
|
169
|
Cot1-546
|
22.9
|
0.8
|
0.5
|
1.7
|
87
|
The results for PPRL are not very good, but it'll have to do. I should probably add more sample during hybridization.
Sample Matrix
Sample
|
uL PPRL-488 (pmol)
|
uL Cot1-546 (pmol)
|
uL DMD-546 (pmol)
|
uL Cot1-488 (ug)
|
A
|
25 (25)
|
25 (0.6)
|
0
|
0
|
B
|
0
|
0
|
10.5 (40)
|
6 (0.3)
|
- Allow stored samples to warm to room temperature
- Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
- Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
- Remove samples and allow to cool to room temperature
- Add 25 uL hybridization cocktail to center
- Add 22x22 #1.5 coverslip; seal with rubber cement
- Allow the rubber cement to air-dry for 5 minutes at room temperature
- Denature for 2.5 minutes at 92C; keep humid
- Transfer slides to a humidified chamber and hybridize overnight at 37C