Daniel:Notebook/HiResChrPaint/2014-1-24: Difference between revisions
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>Djacobse |
>Djacobse (→FISH) |
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(3 intermediate revisions by the same user not shown) | |||
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'''Samples''': | '''Samples''': | ||
*5 ug Cot1 DNA | *5 ug Cot1 DNA | ||
*The entire sample of [[Daniel:Notebook/HiResChrPaint/2014-1-13#Nanodrop Results]] | *The entire sample of [[Daniel:Notebook/HiResChrPaint/2014-1-13#Nanodrop Results|V6S3-B]] | ||
===Protocol=== | |||
*[[Daniel:Protocols/AlexaDyeCouple#ULYSIS|Following ULYSIS protocol]] | *[[Daniel:Protocols/AlexaDyeCouple#ULYSIS|Following ULYSIS protocol]] | ||
Line 22: | Line 24: | ||
5-1 Measure pre-column concentration using nanodrop | 5-1 Measure pre-column concentration using nanodrop | ||
6. Purify using a [[Daniel:Protocols/CentriSep|Centri-Sep column]] | 6. Purify using a [[Daniel:Protocols/CentriSep|Centri-Sep column]] | ||
===Labeling Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| align="center" width="65" height="15" | | |||
|style="background-color:#8DB4E2;font-weight:bold" width="65" colspan="4" | Pre Column | |||
|style="background-color:#E6B8B7;font-weight:bold" width="65" colspan="4" | Post Column | |||
| align="center" width="71" | | |||
| align="center" width="65" | | |||
|- style="font-size:12pt;font-weight:bold" align="center" | |||
|style="background-color:#D9D9D9" height="45" | Sample | |||
|style="background-color:#8DB4E2" | ng/uL ssDNA | |||
|style="background-color:#8DB4E2" | pmol/uL dye | |||
|style="background-color:#8DB4E2" | pmol/uL ss probe | |||
|style="background-color:#8DB4E2" | base:dye | |||
|style="background-color:#E6B8B7" | ng/uL ssDNA | |||
|style="background-color:#E6B8B7" | pmol/uL dye | |||
|style="background-color:#E6B8B7" | pmol/uL ss probe | |||
|style="background-color:#E6B8B7" | base:dye | |||
|style="background-color:#D8E4BC" | DNA retention (%) | |||
|style="background-color:#D8E4BC" | Dye retention (%) | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#D9D9D9;font-weight:bold" height="15" | Cot1 | |||
|style="background-color:#8DB4E2" align="center" | 145.2 | |||
|style="background-color:#8DB4E2" align="center" | 36.7 | |||
|style="background-color:#8DB4E2" align="center" | 2.5 | |||
|style="background-color:#8DB4E2" align="center" | 12 | |||
|style="background-color:#E6B8B7" align="center" | 80.5 | |||
|style="background-color:#E6B8B7" align="center" | 4.6 | |||
|style="background-color:#E6B8B7" align="center" | 1.4 | |||
|style="background-color:#E6B8B7" align="center" | 53 | |||
|style="background-color:#D8E4BC" align="center" | 44.35 | |||
|style="background-color:#D8E4BC" align="center" | 10.03 | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#D9D9D9;font-weight:bold" height="15" | V6S3 | |||
|style="background-color:#8DB4E2" align="center" | 76.4 | |||
|style="background-color:#8DB4E2" align="center" | 24.6 | |||
|style="background-color:#8DB4E2" align="center" | 2.9 | |||
|style="background-color:#8DB4E2" align="center" | 9 | |||
|style="background-color:#E6B8B7" align="center" | 57.7 | |||
|style="background-color:#E6B8B7" align="center" | 0.9 | |||
|style="background-color:#E6B8B7" align="center" | 2.2 | |||
|style="background-color:#E6B8B7" align="center" | 194 | |||
|style="background-color:#D8E4BC" align="center" | 60.42 | |||
|style="background-color:#D8E4BC" align="center" | 2.93 | |||
|} | |||
These are great results, and I now have plenty of DNA for FISH. | |||
=FISH= | |||
Time to try FISH on the positive control. | |||
'''Samples''': | |||
*6 uL labeled Cot1 DNA (above) | |||
*10 uL V6S3 (above) | |||
''Original Protocol from Beliveau et al'': [[Media:Interphase_FISH_protocol.pdf|FISH Protocol (Beliveau)]] | |||
''Day 1'' | |||
0. Dry samples in vacuum centrifuge | |||
1. Allow stored slides 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 slides and allow to cool to room temperature | |||
5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip | |||
6. Invert slides on to cocktail-covered coverslips; 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 |
Latest revision as of 22:56, 24 January 2014
Alu Control (Started 01/08/2014)[edit]
Cot1 Labeling[edit]
Labeling lots of Cot1 DNA for FISH experiments.
Samples:
- 5 ug Cot1 DNA
- The entire sample of V6S3-B
Protocol[edit]
1. 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! 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 5-1 Measure pre-column concentration using nanodrop 6. Purify using a Centri-Sep column
Labeling Results[edit]
Pre Column | Post Column | |||||||||
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ss probe | base:dye | ng/uL ssDNA | pmol/uL dye | pmol/uL ss probe | base:dye | DNA retention (%) | Dye retention (%) |
Cot1 | 145.2 | 36.7 | 2.5 | 12 | 80.5 | 4.6 | 1.4 | 53 | 44.35 | 10.03 |
V6S3 | 76.4 | 24.6 | 2.9 | 9 | 57.7 | 0.9 | 2.2 | 194 | 60.42 | 2.93 |
These are great results, and I now have plenty of DNA for FISH.
FISH[edit]
Time to try FISH on the positive control.
Samples:
- 6 uL labeled Cot1 DNA (above)
- 10 uL V6S3 (above)
Original Protocol from Beliveau et al: FISH Protocol (Beliveau)
Day 1
0. Dry samples in vacuum centrifuge 1. Allow stored slides 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 slides and allow to cool to room temperature 5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip 6. Invert slides on to cocktail-covered coverslips; 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