Daniel:Notebook/PosSequencing/2017-6-2

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Emulsion PCR Test 3 (Started Wednesday)[edit]

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Protocol-Part 2[edit]

  1. Single Primer qPCR
    1. Make the following master mixes
    2. Reagent uL in Single Rxn Master Mix P2 (5.2X) Master Mix BioU (5.2X) Master Mix Dual (5.2X)
      Primer 2 (10 uM) 1 5.2 0 5.2
      BioU Primer (10 uM) 1 0 5.2 5.2
      2X Kapa SYBR Master Mix 20 104 104 104
      nfH2O 17 88.4 88.4 83.2
      Total* (+ 2 uL sample=40) 38 197.6 197.6 197.6
    3. Aliquot 38 uL master mix to appropriate lanes
    4. Add 2 uL sample according to plate layout below
    5. File:PlateLayout-20170601-SPqPCR-emPCRTest3-Round2.png
    6. Run the following thermocycler program
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x120
      7. 72C 2 min
      8. 16C hold

Results[edit]

Conclusions:

  • It didn't show well, but the H4 lane (dsDNA +++ control, dual primer) had a weird curve that started negative and went to zero quickly. It could have been poor baseline correction (platewise instead of by lane or something), in which case it actually CT'd 1st.
  • There is just too little sample left (if there is any at all) for detection. Even the dual primers never really amplified. I could modify the protocol to save more along the way, or test other methods

Aqueous PCR Test[edit]

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The general purpose of this test is to test the efficacy of the emPCR protocol (minus the emulsion). I will do the aqueous reaction and subject it to the emPCR thermocycler protocol. Then I'll pull down the beads and remove the supernatant (but save it). Perform a couple of washes, saving the wash, and then finish with the final fraction. This should give me an idea of the effects of temperature on the reaction. If the experiment shows plenty of DNA left at that point then it is something to do with the emulsion conditions, and the PCR protocol is fine (although reagent concentrations may need to be adjusted).

Protocol[edit]

  1. Template-Bead Binding
    1. Suspend 2.5 uL (25 ug, ~25M 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 5 uL 10 uM Biotin-Uracil primer1 (50 pmol total, 2X excess) and 15 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 5 uL TE buffer
    8. Mix together the three suspensions of beads with different oligos
    9. Mix ingredients using the following table
    10. Component Ion Torrent Concentration My uL Added- Sample (Single) My Final Concentration
      DreamTaq 10X PCR Buffer (20 mM MgCl2) 1X 5 1X
      10 mM dNTPs 2.5 mM 12.5 2.5 mM
      25 mM MgCl2 3.5 mM 3 3.5 mM
      100 uM Primer A (Free primer-Primer 2) 9 uM 2.5 5 uM
      10 uM Primer B (Bound primer-Primer Biotin-Uracil) 0.1 uM 0.5 0.1 uM
      Hydrogel beads, 2M/uL 50 Million 5 60 M
      DreamTaq Polymerase (5 U/uL) * 90 Units/150 uL 9 90 Units
      DNA template 2nd Dilution (Barcode Oligo v2 1 pM) 0.12 pM 5 0.012 fmol total (0.006 fmol each?)
      1 M Ammonium Sulfate 5 mM 0 0
      1 mM Spermidine*** 100 uM 5 0
      TIPP NA 0 0
      Water NA 2.5 NA
      Total Volume   50  
  2. Emulsion PCR
    1. Perform PCR with the following settings
      1. 95C 2 min
      2. 95C 20 sec
      3. 58C 30 sec
      4. 70C 30 sec
      5. Goto b 40 times
      6. 95C 30 sec
      7. 68C 3 min
      8. Goto f 20X times
      9. 4C forever

Continued tomorrow