Weiss Lab:DNA labeling

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Note: Labeling procedures might be optimized for particular dyes. Check manufacturer instructions. Materials:

Materials

Sodium Bicarbonate (pH 8.3, but acceptable range is 8.0 and 8.8): Na(CO3)2 Sodium Borate (pH 8.3) Acetonitrile: ACN Sodium Acetate (pH 5.2) TE: 10 mM TRIS (pH 8.0) + 1 mM EDTA

Procedure

  1. Suspend DNA from IDT in 100 uL of 50 mM TEAA + 5% ACN
    1. To help dissolve, raise the temperature for 10 mins if necessary
  2. Spin down, pellet will form powdery-crystal residue which is the particulate from the sample that is not DNA. Keep it just in case, since there might be some DNA in that pellet.
  3. HPLC to separate lengths (use Reverse Phase/Size Exclusion column)
    1. There will be one large fraction which is the fraction of interest
  4. Dry the elution/fraction in SpeedVac. After drying, suspend dried fraction in MilliQ water. Check absorbance to determine concentration in UV/Vis Spectrometer.
  5. For labeling the complement strand with ATTO 647, resuspend 100 mM (pH 8.3) Sodium Bicarbonate. For labeling template strand with Cy3B, use 100 mM (pH 8.3) Sodium Borate. Determine concentration of dyes in DMSO using absorbance.

Add dye in excess (10x recommended). Dissolve the dye in anhydrous DMSO/DMF, prepared immediately before labeling reaction. Leave for 24-48 hrs at room temperature.

  1. Ethanol Precipitate. Add 150 mM Sodium Acetate and 2x volume of ethanol
    1. For a 100 uL solution, add 5 uL of Sodium Acetate and 250 uL of ethanol

Place in (-80 or -20) freezer at least 1 hr, overnight is fine. Remove supernatant and make sure all ethanol has evaporated off. #Resuspend in 50 mM TEAA + 5% ACN

  1. HPLC and collect unlabeled peak, labeled peak
  2. Dry in SpeedVacFor long-term storage, leave in TE. Add BSA to prevent sticking if you expect very long-term storage. 
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