Daniel:Protocols/AlexaDyeCouple: Difference between revisions
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==ARES== | ==ARES== | ||
ARES Manual from Invitrogen: [[ | ARES Manual from Invitrogen: [[Media:ARESTM DNA Labeling Kits.pdf|Invitrogen Protocol]] | ||
#Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature | |||
#Add 2.5 uL DNA and 1.5 uL of sodium bicarbonate buffer | |||
#Denature samples for 5 minutes at 95C, then snap cool using ice box | |||
#Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds | |||
##dye cannot be saved for later use. Use immediately! | |||
#Add 1 uL dye to sample | |||
#Incubate in the dark for 1 hour | |||
#Add 5 uL 3M NaOAc and 40 uL nfH20 to sample | |||
#[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation | |||
==ULYSIS== | ==ULYSIS== | ||
ULYSIS Manual from Invitrogen: [[ | ULYSIS Manual from Invitrogen: [[Media:ULYSIS_Manual.pdf|ULYSIS Manual]] | ||
ULYSIS Poster from Molecular Probe: [[File:ULS-ASBMB-2001.pdf]] | ULYSIS Poster from Molecular Probe: [[File:ULS-ASBMB-2001.pdf]] | ||
#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) | |||
[[Image:ULYSIS_ReactionTable.png|500px]] | [[Image:ULYSIS_ReactionTable.png|500px]] |
Latest revision as of 18:18, 24 February 2015
Alexa Fluorophore Dye Coupling[edit]
Dye Couples[edit]
Probe | Alexa Dye |
V4S1 | Alexa-546 |
V4S2 | Alexa-488 |
V4S3 | Alexa-594 |
V4S4 | Alexa-647 |
V6S1 | Alexa-546 |
V6S2 | Alexa-488 |
V6S3 | Alexa-594 |
V6S4 | Alexa-647 |
ARES[edit]
ARES Manual from Invitrogen: Invitrogen Protocol
- Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
- Add 2.5 uL DNA and 1.5 uL of sodium bicarbonate buffer
- Denature samples for 5 minutes at 95C, then snap cool using ice box
- Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
- dye cannot be saved for later use. Use immediately!
- Add 1 uL dye to sample
- Incubate in the dark for 1 hour
- Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
- Centri-Sep column purification after incubation
ULYSIS[edit]
ULYSIS Manual from Invitrogen: ULYSIS Manual
ULYSIS Poster from Molecular Probe: File:ULS-ASBMB-2001.pdf
- 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