Daniel:Notebook/HiResChrPaint/2014-3-6: Difference between revisions
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*Following [[Daniel:Protocols/AlexaDyeCouple#ULYSIS|ULYSIS protocol]] | *Following [[Daniel:Protocols/AlexaDyeCouple#ULYSIS|ULYSIS protocol]] | ||
#Dry samples in vacuum centrifuge | |||
#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) | ||
##For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C) | ##For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C) | ||
Line 47: | Line 48: | ||
[[Image:ULYSIS_ReactionTable.png|500px]] | [[Image:ULYSIS_ReactionTable.png|500px]] | ||
===Labeling Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt;font-weight:bold" align="center" valign="bottom" | |||
| width="65" height="30" | Sample | |||
| width="54" | ng/uL dsDNA | |||
| width="58" | pmol/uL dye | |||
| width="58" | Length (bp) | |||
| width="63" | pmol/uL probe | |||
| width="65" | dye/probe | |||
| width="65" | bp:dye | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S2 | |||
| align="center" | 260.9 | |||
| align="center" | 2.3 | |||
| align="center" | 81 | |||
| align="center" | 4.9 | |||
| align="center" | 0.5 | |||
| align="center" | 172 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Cot1 | |||
| align="center" | 137.5 | |||
| align="center" | 2.9 | |||
| align="center" | 400 | |||
| align="center" | 0.5 | |||
| align="center" | 5.6 | |||
| align="center" | 15 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S3 | |||
| align="center" | 66.1 | |||
| align="center" | 1.7 | |||
| align="center" | 81 | |||
| align="center" | 1.2 | |||
| align="center" | 1.4 | |||
| align="center" | 59 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | BSPP1 | |||
| align="center" | 83.9 | |||
| align="center" | 0.8 | |||
| align="center" | 100 | |||
| align="center" | 1.3 | |||
| align="center" | 0.6 | |||
| align="center" | 129 | |||
|} | |||
[[Category:HRCP]] [[Category:20140305]] [[Category:ULYSIS Label]] |
Latest revision as of 18:06, 2 July 2014
Overload FISH (Started 3/5/2014)[edit]
Dye Labeling[edit]
Samples:
Sample | Amount | Dye |
V6S2(A+B) | 6.3 ug | 488 |
V6S3(A+B) | 6.9 ug | 546 |
Cot1 | 6uL = 6ug | 488 |
BSPP1B | 2.7 ug | 546 |
- Following ULYSIS protocol
- Dry samples in vacuum centrifuge
- 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 dsDNA | pmol/uL dye | Length (bp) | pmol/uL probe | dye/probe | bp:dye |
V6S2 | 260.9 | 2.3 | 81 | 4.9 | 0.5 | 172 |
Cot1 | 137.5 | 2.9 | 400 | 0.5 | 5.6 | 15 |
V6S3 | 66.1 | 1.7 | 81 | 1.2 | 1.4 | 59 |
BSPP1 | 83.9 | 0.8 | 100 | 1.3 | 0.6 | 129 |