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

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<li>Oil Production</li>
<li>Oil Production</li>
<ol type="A">
<ol type="A">
<li>Start from [[Daniel:Notebook/PosSequencing/2017-4-10|Monday April 14]]'s oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)</li></ol>
<li>Start from [[Daniel:Notebook/PosSequencing/2017-4-10|Monday April 10]]'s oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)</li></ol>
<li>Template-Bead Binding; Make 4 samples and follow this protocol for each</li>
<li>Template-Bead Binding; Make 4 samples and follow this protocol for each</li>
<ol type="A">
<ol type="A">
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</ol></ol>
</ol></ol>
</ol>
</ol>
Continued [[Daniel:Notebook/PosSequencing/2017-5-30|Tuesday, May 30]]


=Barcode Oligo Production (Started [[Daniel:Notebook/PosSequencing/2017-5-24|Yesterday]])=
=Barcode Oligo Production (Started [[Daniel:Notebook/PosSequencing/2017-5-24|Yesterday]])=
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</ol></ol>
</ol></ol>


===Results===
==Results==


{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
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| width="190" height="45" | Sample
| width="190" height="45" | Sample
| width="85" | Qubit Conc (ug/mL)
| width="85" | Qubit Conc (ug/mL)
| width="85" | Amount (nM)
| width="100" | Amount (nM)
| width="85" | Total Amount (fmol)
| width="95" | Total Amount (fmol)
| width="85" | Amplification (Over 40 amol)
| width="95" | Amplification (Over 40 amol)


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Barcode Oligo v1
| height="15"  valign="bottom" | Barcode Oligo v1 (EcoRI)
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" align="center" | 33.0
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" align="center" | 526.3
| align="center" align="center" valign="bottom" |  
| align="center" align="center" | 3.16E+04
| align="center" align="center" valign="bottom" |  
| align="center" align="center" | 7.89E+08


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Barcode Oligo v2
| height="15"  valign="bottom" | Barcode Oligo v2 (EcoRI)
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" align="center" | 20.0
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" align="center" | 319.0
| align="center" align="center" valign="bottom" |  
| align="center" align="center" | 2.87E+04
| align="center" align="center" valign="bottom" |  
| align="center" align="center" | 7.18E+08


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Barcode Oligo v3
| height="15"  valign="bottom" | Barcode Oligo v3 (EcoRI)
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" align="center" | 33.7
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" align="center" | 537.5
| align="center" align="center" valign="bottom" |  
| align="center" align="center" | 3.22E+04
| align="center" align="center" valign="bottom" |  
| align="center" align="center" | 8.06E+08
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Barcode Oligo v1 (Uracil)
| align="center" align="center" | 26.3
| align="center" align="center" | 419.5
| align="center" align="center" | 2.52E+04
| align="center" align="center" | 6.29E+08


|}
|- style="font-size:12pt"
| height="15"  valign="bottom" | Barcode Oligo v2 (Uracil)
| align="center" align="center" | 32.6
| align="center" align="center" | 519.9
| align="center" align="center" | 3.12E+04
| align="center" align="center" | 7.80E+08


|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Barcode Oligo v3 (Uracil)
| align="center" align="center" | 33.6
| align="center" align="center" | 535.9
| align="center" align="center" | 3.22E+04
| align="center" align="center" | 8.04E+08


|}


<gallery perrow=2 heights=250px widths=250px mode=packed-hover>
<gallery perrow=2 heights=250px widths=250px mode=packed-hover>
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File:2017-05-25-PosSeq-BarcProduction-Uracil.png|Uracil samples
File:2017-05-25-PosSeq-BarcProduction-Uracil.png|Uracil samples
</gallery>
</gallery>
[[Category:20170524]] [[Category:20170525]] [[Category:OligoPrep]] [[Category:PosSeq]]

Latest revision as of 19:55, 31 May 2017

Emulsion PCR Test 2[edit]

Back to Calendar

From the results on

Protocol[edit]

  1. Oil Production
    1. Start from Monday April 10'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

Continued Tuesday, May 30

Barcode Oligo Production (Started Yesterday)[edit]

Protocol[edit]

  1. Qiaquick Column
  2. Note: I will be pooling samples and double loading columns, yielding 4 samples per column (2 columns per barcode oligo, 12 columns total)
    1. Pool 4 samples and 1000 uL PB (5X binding buffer)
    2. Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
    3. Add another 600 uL pooled sample to columnand spin for 1 minute at 14000 rpm; discard flow through
    4. Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
    5. Dry spin column for 1 minute at 14000 rpm; discard flow through
    6. Let stand with cover open in fume hood for ~5 minutes
    7. Transfer column to a new 1.5 mL eppendorf tube
    8. Add 35 uL nfH2O to column
    9. Let stand 1 minute
    10. Spin for 1 minute at 14000 rpm
    11. Pool samples when finished; concentrate if desired
  3. TBE Gel
    1. Mix 160 uL TBE and 40 uL 6X dye
    2. Aliquot 10 uL mix to parafilm
    3. Add 2 uL sample or 1.5 uL ladder to appropriate aliquot
    4. Add 10 uL mix to appropriate lane
    5. Run gel at 240V for 24 minutes
    6. Open gel and stain with 2 uL SYBR gold for 3 minutes
    7. Rinse and image in gel doc
  4. Qubit
    1. Mix 1194 uL dsDNA buffer and 6 uL dye
    2. Aliquot 190 uL to standard tubes and 199 uL to dye tubes
    3. Add 10 uL standard or 1 uL dye
    4. Vortex and spin down
    5. Let sit 5 minutes in the dark
    6. Measure in the Qubit

Results[edit]

Sample Qubit Conc (ug/mL) Amount (nM) Total Amount (fmol) Amplification (Over 40 amol)
Barcode Oligo v1 (EcoRI) 33.0 526.3 3.16E+04 7.89E+08
Barcode Oligo v2 (EcoRI) 20.0 319.0 2.87E+04 7.18E+08
Barcode Oligo v3 (EcoRI) 33.7 537.5 3.22E+04 8.06E+08
Barcode Oligo v1 (Uracil) 26.3 419.5 2.52E+04 6.29E+08
Barcode Oligo v2 (Uracil) 32.6 519.9 3.12E+04 7.80E+08
Barcode Oligo v3 (Uracil) 33.6 535.9 3.22E+04 8.04E+08