Daniel:Notebook/PosSequencing/2017-7-12: Difference between revisions

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<li>Add 15 uL to a slide for analysis</li>
<li>Add 15 uL to a slide for analysis</li>
<li>Seal coverslip with nail polish and wait approximately 30 minutes to dry</li>
<li>Seal coverslip with nail polish and wait approximately 30 minutes to dry</li>
</ol>
</ol>
</ol>


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</gallery>
</gallery>


[[Category:PosSeq]] [[Category:20170706]]
=emPCR 9-Concentration Comparison=
 
The beads experiment above seemed to work, but I think the beads were a little under-loaded. I'm going to jack up the concentrations to make a new one. Hopefully this will give a bigger gradient of bead percentage loaded.
 
==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 '''3''' 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="263" height="45" | Component
| width="65" | uL Added
| width="65" | 3.2X Master Mix uL Added
| width="87" | Final Concentration
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | DreamTaq 10X PCR Buffer (20 mM MgCl2)
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 32
| align="center" valign="bottom" | 1X
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 10 mM dNTPs
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 32
| align="center" valign="bottom" | 1 mM
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | 25 mM MgCl2
| align="center" align="center" valign="bottom" | 6
| align="center" align="center" valign="bottom" | 19.2
| 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="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 32
| align="center" valign="bottom" | 1 uM
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | 10 uM dual biotin primer
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 1.6
| align="center" valign="bottom" | 0.1 uM
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Hydrogel beads, 2M/uL
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 32
| align="center" valign="bottom" | 25 M
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | DreamTaq Polymerase (5 U/uL) *
| align="center" align="center" valign="bottom" | 3
| align="center" align="center" valign="bottom" | 0
| align="center" valign="bottom" | 15 Units
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="30"  | Barcode Oligo v1 or v3 (template, 100 pM, 1 nM 10 nM)
| align="center" align="center" | 10
| align="center" align="center" | 0
| align="center" | 10 pM, 100 pM, 1 nM
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | 100 mM Ammonium Sulfate
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 16
| align="center" valign="bottom" | 5 mM
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 1 mM Spermidine
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 32
| align="center" valign="bottom" | 100 uM
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Water
| align="center" align="center" valign="bottom" | 25.5
| align="center" align="center" valign="bottom" | 81.6
| align="center" valign="bottom" | NA
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Total Volume
|style="font-weight:bold" align="center" align="center" valign="bottom" | 100
|style="font-weight:bold" align="center" align="center" valign="bottom" | 278.4
| align="center" align="center" valign="bottom" | &nbsp;
 
|}
 
<li>Add 2 uL DreamTaq Polymerase to each sample</li>
</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>45C 30 sec</li>
<li>70C 30 sec</li>
<li>Goto b 95 times</li>
<li>4C forever</li>
</ol></ol>
 
Continued [[Daniel:Notebook/PosSequencing/2017-7-13|tomorrow]]
 
[[Category:PosSeq]] [[Category:20170706]] [[Category:20170712]]

Latest revision as of 23:11, 12 July 2017

emPCR8 (Started Thursday July 6)[edit]

Back to Calendar

Protocol (From Yesterday)[edit]

  1. Dye hybridization
    1. Mix 20 uL ssDNA (lambda digested) and 20 uL 4X SSC buffer
    2. Incubate in the dark at 70C for 5 minutes
    3. Incubate at 55C for 20 minutes
    4. Add 1 uL appropriate (10 uM) dye, A488-dc6 for sample 1 and Cy5-dc6 for sample 3
    5. Incubate in the dark (RT) for 15 minutes
    6. Magnet pulldown, remove supernatant and add 40 uL 2X SSC buffer
    7. Add 15 uL to a slide for analysis
    8. Seal coverslip with nail polish and wait approximately 30 minutes to dry

Results[edit]

emPCR 9-Concentration Comparison[edit]

The beads experiment above seemed to work, but I think the beads were a little under-loaded. I'm going to jack up the concentrations to make a new one. Hopefully this will give a bigger gradient of bead percentage loaded.

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 3 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 3.2X Master Mix uL Added Final Concentration
      DreamTaq 10X PCR Buffer (20 mM MgCl2) 10 32 1X
      10 mM dNTPs 10 32 1 mM
      25 mM MgCl2 6 19.2 3.5 mM
      10 uM Primer A (Free primer-Primer 2) 10 32 1 uM
      10 uM dual biotin primer 0.5 1.6 0.1 uM
      Hydrogel beads, 2M/uL 10 32 25 M
      DreamTaq Polymerase (5 U/uL) * 3 0 15 Units
      Barcode Oligo v1 or v3 (template, 100 pM, 1 nM 10 nM) 10 0 10 pM, 100 pM, 1 nM
      100 mM Ammonium Sulfate 5 16 5 mM
      1 mM Spermidine 10 32 100 uM
      Water 25.5 81.6 NA
      Total Volume 100 278.4  
    6. Add 2 uL DreamTaq Polymerase to each sample
  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. 45C 30 sec
      4. 70C 30 sec
      5. Goto b 95 times
      6. 4C forever

    Continued tomorrow