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  • Downstream Analysis (E2600)

    Protocol

    1. Quantitation of DNA by qPCR (for control fragmented Hela DNA)

      After magnetic capture, aliquot 10 μl each of the supernatant in triplicate for each methylated, unmethylated and control sample.

    2. For input control and saved supernatant of the non-captured DNA fraction, dilute 1 μl of these samples in 1 ml water, aliquot 10 μl each of the sample in triplicate for each methylated, unmethylated and control sample.

    3. Add 1 μl of the primer mix specific to each control locus to each tube.

    4. Add 10 μl of the desired qPCR Master Mix according to manufacturer's recommendations.

    5. Perform qPCR standard reaction program using an annealing temperature of 60°C.

    6. Analyze quantitative PCR results using software provided with the real-time PCR machine.

      At least a 500–1000 fold enrichment of the methylated locus (LINE) versus the active gene (RPL30) within the methylated eluant sample as compared to the input DNA should be observed. Conversely, no enrichment of the methylated locus should be observed in the saved supernatant of the non-captured DNA fraction.

    7. Quantitation of DNA by Standard PCR Reaction (for control fragmented DNA) 

      Perform capture experiment as described previously. Elute sample by incubation at 65°C in 50 μl of DNase-free water. Prepare a master reaction mix as described below:
      Reagent Volume for
      1 PCR Reaction (20 μl)
      DNA Samples (or 1:1000 diluted input DNA) 13.5 μl
      Standard Taq Buffer (10X) 2.0 μl
      dNTP Mix (4 mM) 1.0 μl
      Primers (MyoD, RPL30, MirA, 10 μM) 1.0 μl
      Taq DNA Polymerase 0.5 μl

      Transfer PCR tubes to a thermocycler: 
      Thermocycling conditions for a routine PCR:
      Initial denaturation:
      95°C  5 seconds 
      25 cycles:
      95°C  15 seconds
      60°C  15 seconds
      72°C  30 seconds
      Final extension:
      72°C  5 minutes
      Hold:
      4°C

      Remove 10 μl of each PCR product for analysis by 2% agarose gel or 10% polyacrylamide gel electrophoresis using the 50 bp DNA Ladder (NEB #N3236 ) as a standard. The expected size of all the amplicons is ~75 bp. Alternatively, the eluted sample DNA from the qPCR reaction tube may be used.