Daniel:Notebook/PosSequencing/2017-5-5

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Revision as of 20:38, 5 May 2017 by >Djacobse (→‎Protocol)
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Probe Production

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Dr Zhang had a good suggestion to do biotin probe production for each of the barcode oligos. I'll do this as a standard reaction without the bead oligos. I can then do the biotin attachment and oligo melting after that. This will allow me to separate the imaging steps from the actual emulsion PCR which will be more important for

Protocol

  1. qPCR
    1. Make 4.2X (1X) master mix with the following reagents
      1. 4.2 (1) uL 10 uM AP1v4U
      2. 4.2 (1) uL 10 uM BiotinU
      3. 83.2 (18) uL nfH2O
      4. 104 (25) uL 2X SYBR Fast
    2. Add 45 uL master mix to each sample
    3. Add 5 uL barcode oligo according to plate layout
    4. [[Image:|600px]]
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold
  2. Bead Binding
    1. Suspend 5 uL (50 ug, ~50M beads) beads in 100 uL Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)
    4. Add 2 uL 1 uM (10 pmol total, 2X excess) template oligo and 18 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. Resuspend beads in 10 uL TE buffer
    8. Mix together the three suspensions of beads with different oligos
  3. Melting
    1. Withdraw 2XSSC buffer from end of protocol Yesterday
    2. Resuspend in 100μL of TE-Triton 0.1% (TE-T)
    3. Melt off the complementary DNA strand by incubating the sample at 95°C for 3 minutes
    4. Magnet pull-down for 1 minute and remove supernatant
    5. Add 100 μL of TE-T and mix
    6. Repeat wash
  4. Fluorescent Oligo Binding (All samples)
    1. Resuspend beads in 25 uL 4X SSC buffer
    2. Make the following dye hybridization buffer
      1. 8.4 each uL 10 uM dye; dcprobe6-488, dcprobe6-Cy3, dcprobe6-Cy5
      2. 63 uL formamide
      3. 16.8 uL nfH2O
    3. Add 25 uL fluorescent probe in 60% formamide to each sample
    4. Incubate at room temp in the dark for 30 min
    5. Wash twice in 2X SSC
    6. Resuspend in 40 uL 2XSSC or 20 uL 2X SSC (Sample 1)