Daniel:Notebook/HiResChrPaint/2014-7-1: Difference between revisions
Jump to navigation
Jump to search
>Djacobse (Created page with "=FISH w/ 70% Formamide (Started 06/30/2014)= Back to Calendar ==Dye Couple== #Add 100 uL DMSO ...") |
>Djacobse No edit summary |
||
Line 13: | Line 13: | ||
#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) | ||
===Centri-Sep Column=== | |||
#Hydrate in 800 uL ddH20; Allow at least 30 minutes for hydration, be sure to hydrate all the gel | |||
#Remove air bubbles by inverting and sharply tapping the column; stand up column and allow to settle | |||
#Drain the column into a wash tube (200-250 uL will drain) | |||
#Centrifuge at 750xg for 2 minutes (~3000 rpm; removes 300 uL fluid) | |||
#Transfer 20 uL reaction mixture to the top of the gel; do not disturb gel surface | |||
#Place column into collection tube and centrifuge at 750xg for 2 minutes | |||
#Dry sample in vacuum centrifuge |
Revision as of 18:36, 1 July 2014
FISH w/ 70% Formamide (Started 06/30/2014)
Dye Couple
- 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)
Centri-Sep Column
- Hydrate in 800 uL ddH20; Allow at least 30 minutes for hydration, be sure to hydrate all the gel
- Remove air bubbles by inverting and sharply tapping the column; stand up column and allow to settle
- Drain the column into a wash tube (200-250 uL will drain)
- Centrifuge at 750xg for 2 minutes (~3000 rpm; removes 300 uL fluid)
- Transfer 20 uL reaction mixture to the top of the gel; do not disturb gel surface
- Place column into collection tube and centrifuge at 750xg for 2 minutes
- Dry sample in vacuum centrifuge