Daniel:Notebook/HiResChrPaint/2014-7-15: Difference between revisions
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==Dye Labeling== | ==Dye Labeling== | ||
I need to order more ULYSIS 488, so for now I'll just do 2 ug of V6S2 labeled with Alexa 546. | |||
#Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C) | #Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C) | ||
Line 13: | Line 15: | ||
#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 + 1 uL RNase A | |||
#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 >24 hrs at 42C | |||
##Continued [[Daniel:Notebook/HiResChrPaint/2014-7-17|7/17/2014]] | |||
[[Category:HRCP]] [[Category:20140708]] [[Category:ULYSIS Label]] | [[Category:HRCP]] [[Category:20140708]] [[Category:ULYSIS Label]] [[Category:Hybridization]] |
Latest revision as of 17:24, 17 July 2014
Strand Control (Started 07/08/2014)[edit]
Dye Labeling[edit]
I need to order more ULYSIS 488, so for now I'll just do 2 ug of V6S2 labeled with Alexa 546.
- 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)
Labeling Results[edit]
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[edit]
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 |
- 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 + 1 uL RNase A
- 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 >24 hrs at 42C
- Continued 7/17/2014