Daniel:Notebook/PosSequencing/2017-5-25: Difference between revisions

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>Djacobse
>Djacobse
Line 28: Line 28:
| width="85" | Ion Torrent Concentration
| width="85" | Ion Torrent Concentration
| width="110" | My uL Added- Sample (Single)
| width="110" | My uL Added- Sample (Single)
| width="95" | uL Added MM (2.2X)
| width="180" | My Final Concentration
| width="180" | My Final Concentration


Line 35: Line 34:
| align="center" valign="bottom" | 1X
| align="center" valign="bottom" | 1X
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 22
| align="center" valign="bottom" | 1X
| align="center" valign="bottom" | 1X


Line 42: Line 40:
| align="center" valign="bottom" | 2.5 mM
| align="center" valign="bottom" | 2.5 mM
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 55
| align="center" valign="bottom" | 2.5 mM
| align="center" valign="bottom" | 2.5 mM


Line 49: Line 46:
| align="center" valign="bottom" | 3.5 mM
| align="center" valign="bottom" | 3.5 mM
| align="center" align="center" valign="bottom" | 6
| align="center" align="center" valign="bottom" | 6
| align="center" align="center" valign="bottom" | 13.2
| align="center" valign="bottom" | 3.5 mM
| align="center" valign="bottom" | 3.5 mM


Line 56: Line 52:
| align="center" valign="bottom" | 9 uM
| align="center" valign="bottom" | 9 uM
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 11
| align="center" valign="bottom" | 5 uM
| align="center" valign="bottom" | 5 uM


Line 63: Line 58:
| align="center" valign="bottom" | 0.1 uM
| align="center" valign="bottom" | 0.1 uM
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 2.2
| align="center" valign="bottom" | 0.1 uM
| align="center" valign="bottom" | 0.1 uM


Line 70: Line 64:
| align="center" valign="bottom" | 50 Million
| align="center" valign="bottom" | 50 Million
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 22
| align="center" valign="bottom" | 60 M
| align="center" valign="bottom" | 60 M


Line 77: Line 70:
| align="center" valign="bottom" | 90 Units
| align="center" valign="bottom" | 90 Units
| align="center" align="center" valign="bottom" | 18
| align="center" align="center" valign="bottom" | 18
| align="center" align="center" valign="bottom" | 39.6
| align="center" valign="bottom" | 90 Units
| align="center" valign="bottom" | 90 Units


Line 84: Line 76:
| align="center" | 0.12 pM
| align="center" | 0.12 pM
| align="center" align="center" | 10
| align="center" align="center" | 10
| align="center" align="center" | 22
| align="center" valign="bottom" | 0.012 fmol total (0.006 fmol each?)
| align="center" valign="bottom" | 0.012 fmol total (0.006 fmol each?)


Line 90: Line 81:
| height="15"  valign="bottom" | 1 M Ammonium Sulfate
| height="15"  valign="bottom" | 1 M Ammonium Sulfate
| align="center" valign="bottom" | 5 mM
| align="center" valign="bottom" | 5 mM
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
Line 98: Line 88:
| align="center" valign="bottom" | 100 uM
| align="center" valign="bottom" | 100 uM
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 22
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0


Line 104: Line 93:
| height="15"  valign="bottom" | TIPP
| height="15"  valign="bottom" | TIPP
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
Line 112: Line 100:
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 11
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA


Line 119: Line 106:
| align="center" align="center" valign="bottom" |  
| align="center" align="center" valign="bottom" |  
|style="font-weight:bold" align="center" align="center" valign="bottom" | 100
|style="font-weight:bold" align="center" align="center" valign="bottom" | 100
|style="font-weight:bold" align="center" align="center" valign="bottom" | 220
| align="center" align="center" valign="bottom" |  
| align="center" align="center" valign="bottom" |  



Revision as of 16:21, 25 May 2017

Emulsion PCR Test 2

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From the results on

Protocol

  1. Oil Production
    1. Start from Monday April 14's oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)
  2. Template-Bead Binding; Make 4 samples and follow this protocol for each
    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
    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 10 1X
      10 mM dNTPs 2.5 mM 25 2.5 mM
      25 mM MgCl2 3.5 mM 6 3.5 mM
      100 uM Primer A (Free primer-Primer 2) 9 uM 5 5 uM
      10 uM Primer B (Bound primer-Primer 1) 0.1 uM 1 0.1 uM
      Hydrogel beads, 2M/uL 50 Million 10 60 M
      DreamTaq Polymerase (5 U/uL) * 90 Units 18 90 Units
      DNA template 2nd Dilution (Barcode Oligos 1 pM) 0.12 pM 10 0.012 fmol total (0.006 fmol each?)
      1 M Ammonium Sulfate 5 mM 0 0
      1 mM Spermidine*** 100 uM 10 0
      TIPP NA 0 0
      Water NA 5 NA
      Total Volume   100  
    11. Aliquot 90 uL master mix into 4 tubes
    12. Add 10 uL appropriate primers according to sample table
    13. Aliquot 50 uL each non-emulsion sample into new tubes (A/B)
  3. Oil-Aqueous Mixing
    1. Mix 20 uL aqueous into 200 uL oil mixture
    2. Vortex for 30 seconds
    3. Repeat mixing to 100 uL (5 times)
    4. Mix with vortexer for 3 minutes
    5. Dilute with pure mineral oil for imaging (1:100)
    6. Distribute 300 uL total volume into 6 tubes, 50 uL each; use same pipette tip; only made 5 (oil sticks to tip)
  4. 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