Daniel:Notebook/PosSequencing/2017-6-26: Difference between revisions

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>Djacobse
(Created page with "=Beadless emPCR (Started Thursday 6/22/17)= Back to calendar Since the results on Daniel:Note...")
 
>Djacobse
 
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<ol type="A">
<ol type="A">
<li>Make the following master mixes</li>
<li>Make the following master mixes</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center"
| width="210" height="30" | Reagent
| width="95" | uL in Single Rxn
| width="95" | Master Mix P2 (4.2X)
| width="95" | Master Mix BioU (4.2X)
| width="95" | Master Mix Dual (6.2X)
|- style="font-size:12pt"
| height="15"  valign="bottom" | Primer 2 (10 uM)
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 4.2
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 6.2
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | BioU Primer (10 uM)
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 4.2
| align="center" align="center" valign="bottom" | 6.2
|- style="font-size:12pt"
| height="15"  valign="bottom" | 2X Kapa SYBR Master Mix
| align="center" align="center" valign="bottom" | 20
| align="center" align="center" valign="bottom" | 84
| align="center" align="center" valign="bottom" | 84
| align="center" align="center" valign="bottom" | 124
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 16
| align="center" align="center" valign="bottom" | 67.2
| align="center" align="center" valign="bottom" | 67.2
| align="center" align="center" valign="bottom" | 93
|- style="font-size:12pt"
| height="15"  valign="bottom" | Total* (+ 5 uL sample=40)
|style="font-weight:bold" align="center" align="center" valign="bottom" | 35
|style="font-weight:bold" align="center" align="center" valign="bottom" | 155.4
|style="font-weight:bold" align="center" align="center" valign="bottom" | 155.4
|style="font-weight:bold" align="center" align="center" valign="bottom" | 229.4
|}


<li>Aliquot 35 uL master mix to appropriate lanes</li>
<li>Aliquot 35 uL master mix to appropriate lanes</li>
Line 40: Line 85:
</ol></ol>
</ol></ol>
</ol>
</ol>
==Results==
<gallery perrow=2 heights=250px widths=350px mode=packed-hover>
File:PlateLayout-Beadless-emPCR-singleprimer-20170626.png|Plate layout
File:Beadless-emPCR-singleprimer-20170626-plateCTs-normed.png|Plate CTs
File:Beadless-emPCR-singleprimer-20170626-rawcurves.png|Raw curves
</gallery>
=emPCR 6=
Based on the results from [[Daniel:Notebook/PosSequencing/2017-6-23|Friday]] and continued above, I think this means I'm clear to try again on emPCR using the lower annealing temp. I'll give 15 more cycles since the pM sample didn't amplify.
==Protocol==
<ol>
<li>Oil phase</li>
<ol type="A">
<li>Mix 5 uL Triton X, 40 uL Tween-80, and 450 uL Span 80 into 2 mL Mineral oil and 8 mL Tegosoft DEC</li>
<li>Vortex vigorously to mix</li>
</ol>
<li>Template-Bead Binding; Make 4 samples and follow this protocol for each</li>
<ol type="A">
<li>Suspend 2.5 uL (25 ug, ~25M beads) beads in 100 uL Dynabuffer</li>
<li>Apply magnet for 30 sec and remove supernatant</li>
<li>Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)</li>
<li>Add 5 uL 10 uM (25 pmol total, 2X excess) dual biotin oligo and 15 uL nfH<sub>2</sub>O per sample to bead solution; incubate at RT for 15 min</li>
</ol>
<li>Aqueous Phase Preparation</li>
<ol type="A">
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li>
<li>Repeat wash step</li>
<li>Resuspend beads in 10 uL TE buffer</li>
<li>Mix ingredients using the following table</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#CC66FF;font-size:12pt;font-weight:bold" align="center"
| width="290" height="30" | Component
| width="95" | uL Added
| width="95" | uL Added
| width="120" | Final Concentration
|- style="font-size:12pt"
| height="15"  valign="bottom" | DreamTaq 10X PCR Buffer (20 mM MgCl2)
| align="right" align="center" valign="bottom" | 10
| align="right" align="center" valign="bottom" | 10
| align="center" valign="bottom" | 1X
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 10 mM dNTPs
| align="right" align="center" valign="bottom" | 10
| align="right" align="center" valign="bottom" | 10
| align="center" valign="bottom" | 1 mM
|- style="font-size:12pt"
| height="15"  valign="bottom" | 25 mM MgCl2
| align="right" align="center" valign="bottom" | 6
| align="right" align="center" valign="bottom" | 6
| align="center" valign="bottom" | 3.5 mM
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 10 uM Primer A (Free primer-Primer 2)
| align="right" align="center" valign="bottom" | 10
| align="right" align="center" valign="bottom" | 10
| align="center" valign="bottom" | 1 uM
|- style="font-size:12pt"
| height="15"  valign="bottom" | 10 uM dual biotin primer
| align="right" align="center" valign="bottom" | 0.5
| align="right" align="center" valign="bottom" | 0.5
| align="center" valign="bottom" | 0.1 uM
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Hydrogel beads, 2M/uL
| align="right" align="center" valign="bottom" | 10
| align="right" align="center" valign="bottom" | 10
| align="center" valign="bottom" | 25 M
|- style="font-size:12pt"
| height="15"  valign="bottom" | DreamTaq Polymerase (5 U/uL) *
| align="right" align="center" valign="bottom" | 2
| align="right" align="center" valign="bottom" | 2
| align="center" valign="bottom" | 10 Units
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  | Barcode Oligo v1 (template, 100 pM, 100 nM)
| align="right" align="center" | 5
| align="center" | 5*
| align="center" valign="bottom" | 10 pM, 10 nM*
|- style="font-size:12pt"
| height="15"  valign="bottom" | 100 mM Ammonium Sulfate
| align="right" align="center" valign="bottom" | 5
| align="right" align="center" valign="bottom" | 5
| align="center" valign="bottom" | 5 mM
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 1 mM Spermidine***
| align="right" align="center" valign="bottom" | 10
| align="right" align="center" valign="bottom" | 10
| align="center" valign="bottom" | 100 uM
|- style="font-size:12pt"
| height="15"  valign="bottom" | TIPP
| align="right" align="center" valign="bottom" | 0
| align="right" align="center" valign="bottom" | 0
| align="right" align="center" valign="bottom" | 0
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Water
| align="right" align="center" valign="bottom" | 36.5
| align="right" align="center" valign="bottom" | 36.5
| align="center" valign="bottom" | NA
|- style="font-size:12pt"
| height="15"  valign="bottom" | Total Volume
|style="font-weight:bold" align="right" align="center" valign="bottom" | 100
|style="font-weight:bold" align="right" align="center" valign="bottom" | 100
| align="right" align="center" valign="bottom" | &nbsp;
|}
</ol>
<li>Oil-Aqueous Mixing</li>
<ol type="A">
<li>Mix 10 uL aqueous into 100 uL oil mixture</li>
<li>Vortex for 30 seconds</li>
<li>Repeat mixing to 100 uL (5 times)</li>
<li>Mix with vortexer for 3 minutes</li>
<li>Dilute with pure mineral oil for imaging (1:100)</li>
<li>Distribute 150 uL total volume into 3 tubes, 50 uL each; use same pipette tip</li>
</ol>
<li>Emulsion PCR</li>
<ol type="A">
<li>Perform PCR with the following settings</li>
<ol type="a">
<li>95C 2 min</li>
<li>95C 20 sec</li>
<li>58C 30 sec</li>
<li>70C 30 sec</li>
<li>Goto b 95 times</li>
<li>4C forever</li>
</ol></ol>
</ol>
Continued [[Daniel:Notebook/PosSequencing/2017-6-27|tomorrow]]
[[Category:PosSeq]] [[Category:20170622]] [[Category:20170626]]

Latest revision as of 23:35, 26 June 2017

Beadless emPCR (Started Thursday 6/22/17)[edit]

Back to calendar

Since the results on Friday showed viable product (at least in the 100 nM sample), I'm going to finish up that experiment with strand denaturation and single primer qPCR.

Protocol[edit]

  1. Strand Separation (DNA Denaturation)
    1. Add 500 uL KOH buffer (pH 12.0)
    2. Spin down sample at 13,000rpm for 1 minute
    3. Remove all but 40uL buffer
    4. Repeat KOH steps A-C once more
    5. Add 500 uL 4X SSC buffer (pH 12.0)
    6. Spin down sample at 13,000rpm for 1 minute
    7. Remove all but 10uL buffer
    8. Resuspend in 80 uL 2X SSC buffer
  2. Single primer qPCR
    1. Make the following master mixes
    2. Reagent uL in Single Rxn Master Mix P2 (4.2X) Master Mix BioU (4.2X) Master Mix Dual (6.2X)
      Primer 2 (10 uM) 1 4.2 0 6.2
      BioU Primer (10 uM) 1 0 4.2 6.2
      2X Kapa SYBR Master Mix 20 84 84 124
      nfH2O 16 67.2 67.2 93
      Total* (+ 5 uL sample=40) 35 155.4 155.4 229.4
    3. Aliquot 35 uL master mix to appropriate lanes
    4. Add 2 uL sample according to plate layout below
    5. File:PlateLayout-Beadless-emPCR-singleprimer-20170626.png
    6. 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]

emPCR 6[edit]

Based on the results from Friday and continued above, I think this means I'm clear to try again on emPCR using the lower annealing temp. I'll give 15 more cycles since the pM sample didn't amplify.

Protocol[edit]

  1. Oil phase
    1. Mix 5 uL Triton X, 40 uL Tween-80, and 450 uL Span 80 into 2 mL Mineral oil and 8 mL Tegosoft DEC
    2. Vortex vigorously to mix
  2. Template-Bead Binding; Make 4 samples and follow this protocol for each
    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 (25 pmol total, 2X excess) dual biotin oligo and 15 uL nfH2O per sample to bead solution; incubate at RT for 15 min
  3. Aqueous Phase Preparation
    1. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    2. Repeat wash step
    3. Resuspend beads in 10 uL TE buffer
    4. Mix ingredients using the following table
    5. Component uL Added uL Added Final Concentration
      DreamTaq 10X PCR Buffer (20 mM MgCl2) 10 10 1X
      10 mM dNTPs 10 10 1 mM
      25 mM MgCl2 6 6 3.5 mM
      10 uM Primer A (Free primer-Primer 2) 10 10 1 uM
      10 uM dual biotin primer 0.5 0.5 0.1 uM
      Hydrogel beads, 2M/uL 10 10 25 M
      DreamTaq Polymerase (5 U/uL) * 2 2 10 Units
      Barcode Oligo v1 (template, 100 pM, 100 nM) 5 5* 10 pM, 10 nM*
      100 mM Ammonium Sulfate 5 5 5 mM
      1 mM Spermidine*** 10 10 100 uM
      TIPP 0 0 0
      Water 36.5 36.5 NA
      Total Volume 100 100  
  4. Oil-Aqueous Mixing
    1. Mix 10 uL aqueous into 100 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 150 uL total volume into 3 tubes, 50 uL each; use same pipette tip
  5. 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 95 times
      6. 4C forever

Continued tomorrow