Daniel:Notebook/HiResChrPaint/2014-8-10: Difference between revisions

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>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...")
 
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=FISH=
=FISH with DAPI=


[[Daniel:Notebook/HiResChrPaint|Back to Calendar]]
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]]
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'''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
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#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]

Back to Calendar

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

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
  1. Allow stored samples to warm to room temperature
  2. Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
  3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
  4. Remove samples and allow to cool to room temperature
  5. Add 25 uL hybridization cocktail to center
  6. Add 22x22 #1.5 coverslip; seal with rubber cement
  7. Allow the rubber cement to air-dry for 5 minutes at room temperature
  8. Denature for 2.5 minutes at 92C; keep humid
  9. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C