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)
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  [[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]

Back to Calendar

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
  1. Dry samples in vacuum centrifuge
  2. 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!
  3. Resuspend DNA in 20 uL labeling buffer (Component C)
  4. Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
  5. Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
  6. Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
  7. 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