Daniel:Protocols/AlexaDyeCouple: Difference between revisions

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ARES Manual from Invitrogen: [[Media:ARESTM DNA Labeling Kits.pdf|Invitrogen Protocol]]
ARES Manual from Invitrogen: [[Media:ARESTM DNA Labeling Kits.pdf|Invitrogen Protocol]]


1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
#Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
2. Add 2.5 uL DNA and 1.5 uL of sodium bicarbonate buffer
#Add 2.5 uL DNA and 1.5 uL of sodium bicarbonate buffer
3. Denature samples for 5 minutes at 95C, then snap cool using ice box
#Denature samples for 5 minutes at 95C, then snap cool using ice box
4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
#Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
    *dye cannot be saved for later use.  Use immediately!
##dye cannot be saved for later use.  Use immediately!
5. Add 1 uL dye to sample
#Add 1 uL dye to sample
6. Incubate in the dark for 1 hour
#Incubate in the dark for 1 hour
7. Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
#Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
8. Recommend column purification after incubation
#[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation


==ULYSIS==
==ULYSIS==
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ULYSIS Poster from Molecular Probe: [[File:ULS-ASBMB-2001.pdf]]
ULYSIS Poster from Molecular Probe: [[File:ULS-ASBMB-2001.pdf]]


1. 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)
  ***VORTEX THESE SOLUTIONS '''VIGOROUSLY''' BEFORE USE '''EVERY TIME'''!
##VORTEX THESE SOLUTIONS '''VIGOROUSLY''' BEFORE USE '''EVERY TIME'''!
2. Resuspend DNA in 20 uL labeling buffer (Component C)
#Resuspend DNA in 20 uL labeling buffer (Component C)
3. Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
#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
#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
#Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
6. Purify using a column (Centri-Sep recommended)
#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]

Back to Main

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

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
  2. Add 2.5 uL DNA and 1.5 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
    1. dye cannot be saved for later use. Use immediately!
  5. Add 1 uL dye to sample
  6. Incubate in the dark for 1 hour
  7. Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
  8. Centri-Sep column purification after incubation

ULYSIS[edit]

ULYSIS Manual from Invitrogen: ULYSIS Manual

ULYSIS Poster from Molecular Probe: File:ULS-ASBMB-2001.pdf

  1. 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!
  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
  6. Purify using a column (Centri-Sep recommended)
File:ULYSIS ReactionTable.png