Daniel:Notebook/HiResChrPaint/2014-7-15: Difference between revisions

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#Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
#Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
#Purify using a column (Centri-Sep recommended)
#Purify using a column (Centri-Sep recommended)
===Labeling Results===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#BFBFBF;font-size:12pt;font-weight:bold" align="center"
| width="65" height="30" | Sample
| width="65" | ng/uL ssDNA
| width="65" | pmol/uL dye
| width="65" | pmol/uL ssDNA
| width="71" | dye/probe
| width="65" | bp:dye ratio
|- style="font-size:12pt" align="center"
| height="15" | V6S2-546
| align="center" | 60.2
| align="center" | 3.5
| align="center" | 2.3
| align="center" | 1.6
| align="center" | 52
|- style="font-size:12pt" align="center"
| height="15" | V6S3-546
| align="center" | 74.9
| align="center" | 3.9
| align="center" | 2.8
| align="center" | 1.4
| align="center" | 58
|}
Some of my best results yet.  Proceeding right to FISH.
==Hybridization==
'''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="30" | Sample
| width="65" | [[Daniel:Notebook/HiResChrPaint/2014-1-24#Labeling Results|Cot1-488]] (ug)
| width="65" | V6S2-546 (pmol)
| width="65" | V6S3-546 (pmol)
| width="65" | Cot1 (ug)
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | A
| align="center" | 0
| 8.7 (20)
| 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
| 7.1 (20)
| 2.5 (2.5)
|- style="font-size:12pt" align="center" valign="bottom"
|style="font-weight:bold" height="15" | C
| 5 (0.4)
| align="center" | 0
| align="center" | 0
| align="center" | 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




[[Category:HRCP]] [[Category:20140708]] [[Category:ULYSIS Label]]
[[Category:HRCP]] [[Category:20140708]] [[Category:ULYSIS Label]]

Revision as of 23:23, 15 July 2014

Strand Control (Started 07/08/2014)

Back to Calendar

Dye Labeling

I need to order more ULYSIS 488, so for now I'll just do 2 ug of V6S2 labeled with Alexa 546.

  1. Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C)
    1. For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C)
    2. VORTEX THESE SOLUTIONS VIGOROUSLY BEFORE USE EVERY TIME!
  2. Resuspend DNA in 20 uL labeling buffer (Component C)
  3. Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
  4. Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
  5. Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
  6. Purify using a column (Centri-Sep recommended)

Labeling Results

Sample ng/uL ssDNA pmol/uL dye pmol/uL ssDNA dye/probe bp:dye ratio
V6S2-546 60.2 3.5 2.3 1.6 52
V6S3-546 74.9 3.9 2.8 1.4 58

Some of my best results yet. Proceeding right to FISH.

Hybridization

Sample Matrix

Sample Cot1-488 (ug) V6S2-546 (pmol) V6S3-546 (pmol) Cot1 (ug)
A 0 8.7 (20) 0 2.5 (2.5)
B 0 0 7.1 (20) 2.5 (2.5)
C 5 (0.4) 0 0 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