Daniel:Notebook/PosSequencing/2017-5-18

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Bead Binding Test 3-Alkaline Denaturation (Started Yesterday)[edit]

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Before I can do the true alkaline denaturation experiment I want to test the dyes again. DAPI didn't work, nor did SYBR green, but in both cases they are used to measure DNA without washing afterwards (DAPI is usually added right before adding to slide and SYBR green is used during PCR without using any wash steps, just the difference between ssDNA and dsDNA). So we'll try SYBR again minus washing.

Dye Test[edit]

Simple test to determine viability of the SYBR gree dye (from Kapa SYBR Master Mix) for the assay.

  1. Bead Binding
    1. Suspend 1 uL (10 ug, ~5M beads designed at 2.5 pmol) beads in 100 uL Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend in 10 uL Dynabuffer (conc. 1 ug/uL)
    4. Add 5 uL barcode oligo,either PCCT (10 uM, ssDNA) or Barcode Oligo 2, and up to 5 uL nfH2O per sample to bead solution; incubate at RT for 15 min
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    6. Repeat wash step
    7. Add 50 uL Kapa SYBR Fast master mix 2X + nfH2O at a 1:1 ratio; incubate in the dark for 15 minutes;
    8. Add 15 uL to a slide for analysis
    9. Check in microscope

Results[edit]

So there may be a slight difference in brightness between the dsDNA and the ssDNA, but really there is no easily discernable difference, which is what I was hoping for. Judging by yesterday's results, washing the solution with SYBR green in it pretty much removes all the SYBR. So SYBR probably won't work.