Daniel:Notebook/HiResChrPaint/2014-8-10: Difference between revisions
Jump to navigation
Jump to search
>Djacobse (Created page with "=FISH= Back to Calendar ==Slide Preparation== #Prepare a cell suspension of 500 000-2 000 000 cells/mL in RPMI #Add 20 uL of cell suspensi...") |
>Djacobse No edit summary |
||
(3 intermediate revisions by the same user not shown) | |||
Line 1: | Line 1: | ||
=FISH= | =FISH with DAPI= | ||
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | ||
Line 19: | Line 19: | ||
'''Sample Matrix''' | '''Sample Matrix''' | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="45" | Sample | |||
| width="65" | Cot1-488 (below) (ug) | |||
| width="65" | Dystrophin-488 (pmol) | |||
| width="65" | Dystrophin-546 (pmol) | |||
| width="65" | Cot1 (ug) | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | A | |||
| align="center" | 0 | |||
| 8.6 (30) | |||
| align="center" | 0 | |||
| 2.5 (2.5) | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | B | |||
| align="center" | 0 | |||
| align="center" | 0 | |||
| 7.9 (30) | |||
| 2.5 (2.5) | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | C | |||
| 8.5 (0.4) | |||
| align="center" | 0 | |||
| align="center" | 0 | |||
| align="center" | 0 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" valign="bottom" | |||
|style="font-weight:bold" height="15" | D | |||
| 8.5 (0.4) | |||
| 0 | |||
| align="center" | 7.9 (30) | |||
| align="center" | 0 | |||
|} | |||
#Allow stored samples to warm to room temperature | #Allow stored samples to warm to room temperature | ||
Line 29: | Line 67: | ||
#Denature for 2.5 minutes at 92C; keep humid | #Denature for 2.5 minutes at 92C; keep humid | ||
#Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C | #Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C | ||
[[Category:HRCP]] [[Category:Hybridization]] [[Category:08102014]] |
Latest revision as of 18:51, 12 August 2014
FISH with DAPI[edit]
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
Hybridization[edit]
Sample Matrix
Sample | Cot1-488 (below) (ug) | Dystrophin-488 (pmol) | Dystrophin-546 (pmol) | Cot1 (ug) |
A | 0 | 8.6 (30) | 0 | 2.5 (2.5) |
B | 0 | 0 | 7.9 (30) | 2.5 (2.5) |
C | 8.5 (0.4) | 0 | 0 | 0 |
D | 8.5 (0.4) | 0 | 7.9 (30) | 0 |
- 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 or 42C