Daniel:Notebook/PosSequencing/2017-6-23: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
>Djacobse
 
Line 70: Line 70:
</gallery>
</gallery>


So this shows that it works well. Notice that the nM input (10 nM reaction concentration, 1 pmol 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.
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.


[[Category:PosSeq]] [[Category:20170622]]
[[Category:PosSeq]] [[Category:20170622]]

Latest revision as of 22:43, 23 June 2017

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.