Daniel:Notebook/HiResChrPaint/2013-4-17: Difference between revisions

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==Dye Coupling Test and EtOH Precipitation==
==Dye Coupling Test and EtOH Precipitation==
   Purpose: To test the dye coupling reaction (again), this time purifying post-reaction using ethanol precipitation.  Will take longer (typical suggestion is overnight incubation) but could be more effective than the column preps.
   Purpose: To test the dye coupling reaction (again), this time purifying post-reaction using ethanol precipitation.   
    Will take longer (typical suggestion is overnight incubation) but could be more effective than the column preps.


===Dye Coupling Reaction===
===Dye Coupling Reaction===
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     Chose Alexa dye 546 to match typical color scheme of hi-res project
     Chose Alexa dye 546 to match typical color scheme of hi-res project
   6. Incubate reaction in the dark for 1 hour
   6. Incubate reaction in the dark for 1 hour
  7. Column purify using ssDNA kit from Zymo


===Ethanol Precipitation===
===Ethanol Precipitation===
   1. Add 0.5 (1/10X) uL NaOac, 0.5 uL glycoblue (Recommended: 1/300X but that would be .03) and 30 uL 100% EtOH to each tube (Rec: 2.5X)
   1. Add:
   2. Incubate overnight at -80C
        0.5 uL NaOac (Recommended: 1/10X)
        0.5 uL glycoblue (Recommended: 1/300X but that would be .03) and 30 uL  
        100% EtOH to each tube (Rec: 2.5X)
   2. Transfer to a 1.5mL eppendorf and wrap in tinfoil to protect from light
  3. Incubate overnight at -80C


[[Daniel:Notebook/HiResChrPaint|Back to Calendar]]
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]]

Latest revision as of 22:43, 17 April 2013

Dye Coupling Test and EtOH Precipitation[edit]

 Purpose: To test the dye coupling reaction (again), this time purifying post-reaction using ethanol precipitation.  
   Will take longer (typical suggestion is overnight incubation) but could be more effective than the column preps.

Dye Coupling Reaction[edit]

 1. Thaw out oligos, sodium bicarbonate solution, DMSO
    Use thermo device on Noi's desk to heat DMSO, sodium bicarb to 42 C
 2. Add 2.5 uL DNA, 1.5 uL sodium bicarbonate to 0.2 mL eppendorf
 3. Denature DNA by incubating at 95 C for 5 minutes, followed by snap cooling on ice
 4. Thaw out dyes (they are in big -20C) and resuspend in 2.2 uL DMSO
    Use dyes immediately, they do not store
 5. Add 1 uL of appropriate dye to oligo set (see below)
    Chose V4S2 and V6S2 because of high dna content 
    Chose Alexa dye 546 to match typical color scheme of hi-res project
 6. Incubate reaction in the dark for 1 hour

Ethanol Precipitation[edit]

 1. Add: 
       0.5 uL NaOac (Recommended: 1/10X)
       0.5 uL glycoblue (Recommended: 1/300X but that would be .03) and 30 uL 
       100% EtOH to each tube (Rec: 2.5X)
 2. Transfer to a 1.5mL eppendorf and wrap in tinfoil to protect from light
 3. Incubate overnight at -80C

Back to Calendar