Matt:LabNotes/2015-7-14: Difference between revisions

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(Created page with "=Improve Dye-Coupling= Dan's best practice dye coupling protocol<br> [[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye ...")
 
>Mzcai
mNo edit summary
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[[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye coupling protocol]]
[[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye coupling protocol]]


CUX2-488
CUX2-594
CUX2-594
KIT-488
KIT-488
KIT-594


#Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
#Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
#Add 5 uL DNA (~1ug/ul) and 3 uL of sodium bicarbonate buffer
#Add 5 uL DNA (~1ug/ul) and 3 uL of sodium bicarbonate buffer
#Make 2 25ul aliquots of CUX2 48probe and KIT 48probe to prevent so many freeze-thaws in the future
#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
#Dissolve 1 vial of reactive dye in 2 uL DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing
#Dissolve 1 vial of reactive dye in 2 uL DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing
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#*Use 1X TE Buffer
#*Use 1X TE Buffer
#*No vacufuge
#*No vacufuge
#Repeat column purification
#Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight<!--
#Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight<!--
#Centrifuge at 4 C for 30 minutes (14,000 rpm)
#Centrifuge at 4 C for 30 minutes (14,000 rpm)

Revision as of 18:27, 15 July 2015

Improve Dye-Coupling

Dan's best practice dye coupling protocol
Generic dye coupling protocol

CUX2-594 KIT-488

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
  2. Add 5 uL DNA (~1ug/ul) and 3 uL of sodium bicarbonate buffer
  3. Make 2 25ul aliquots of CUX2 48probe and KIT 48probe to prevent so many freeze-thaws in the future
  4. Denature samples for 5 minutes at 95C, then snap cool using ice box
  5. Dissolve 1 vial of reactive dye in 2 uL DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing
    • Dye cannot be saved for later use. Use immediately!
  6. Add 8 uL sample to dye tube
  7. Incubate in the dark for 1 hour
  8. Add 10 uL 3M NaOAc and 80 uL 1xTE to sample
  9. Centri-Sep column purification after incubation
    • Use 1X TE Buffer
    • No vacufuge
  10. Repeat column purification
  11. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight