Daniel:Notebook/PosSequencing/2017-6-23

From ZhangLabWiki
Jump to navigation Jump to search

Beadless emPCR (Started Yesterday)[edit]

Back to calendar

Protocol[edit]

  1. Prepare Water saturated compounds
    1. Mix 5 mL diethyl ether and 5 mL ddH2O in a falcon tube; allow to settle, extract top (ether) phase
    2. Mix 5 mL ethyl acetate and 5 mL ddH2O in a falcon tube; allow to settle, extract top (acetate) phase
  2. Emulsion Breaking
    1. With a 200 uL pipette add 100 uL diethyl ether to each sample; save the tip
    2. Adjust tip to 200 and using same tip pool samples in a new 1.5 mL tube
    3. Vortex the pooled sample in the 1.5 mL tube for 30 seconds
    4. Spin down the sample 13,000 rpm for 5 minutes
    5. Take off oil-DE mix (top layer) but do not disrupt the oil/aqueous interface
    6. Add 1000 uL ethyl acetate and vortex for 30 seconds
    7. Spin down sample at 13,000rpm for 3 minutes
    8. Remove ethyl acetate without disturbing the pellet
    9. Add 1000 uL diethyl ether and vortex for 30 seconds
    10. Spin down sample at 13,000rpm for 3 minutes
    11. Remove DE without disturbing the aqueous phase
    12. Allow DE to dry in the fume hood
    ******
    
  3. Bead Capture
    1. Add equal volume (100 uL) Dynabuffer wash buffer and 5 uL beads to 1.5 mL tube and vortex for 30 seconds
    2. Incubate at RT for 15 minutes
    3. Pull down with magnet; remove supernatant; Save supernatant for later
    4. Wash twice with 100 uL dynabuffer
    5. Resuspend in 50 uL dynabuffer
    ******
    
  4. qPCR
    1. Make the following 10.2X (1X) master mix
      1. 10.2 (1) uL 10 uM 2Biotin Uracil
      2. 10.2 (1) uL 10 uM Primer 2
      3. 204 (20) uL Kapa SYBR Fast 2X master mix
      4. 163.2 (16) uL nfH2O
    2. Aliquot 38 uL master mix to appropriate lanes
    3. Add 2 uL sample according to plate layout below
    4. File:PlateLayout-20170623-BeadqPCR.png
    5. Run the following thermocycler program
      1. 95C 3 min
      2. 95C 3 sec
      3. 45C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x90
      7. 72C 2 min
      8. 16C hold

Results[edit]

So this shows that it works well. Notice that the nM input (5 nM reaction concentration, 500 fmol total input). Also note that I diluted the beads fraction 1:10 prior to qPCR, which would mean that the initial amount is even higher.