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

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>Djacobse
(Created page with "=Probe Production= Back to Calendar Dr Zhang had a good suggestion to do biotin probe production for each of the barcode oligos. I'll do th...")
 
>Djacobse
 
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<li>qPCR</li>
<li>qPCR</li>
<ol type="A">
<ol type="A">
<li>Make 5.2X (1X) master mix with the following reagents</li>
<li>Make 4.2X (1X) master mix with the following reagents</li>
<ol type="a">
<ol type="a">
<li>4.2 (1) uL 10 uM AP1v4U</li>
<li>4.2 (1) uL 10 uM AP1v4U</li>
<li>4.2 (1) uL 10 uM BiotinU</li>
<li>4.2 (1) uL 10 uM BiotinU</li>
<li>83.2 (18) uL nfH<sub>2</sub>O</li>
<li>75.6 (18) uL nfH<sub>2</sub>O</li>
<li>104 (25) uL 2X SYBR Fast</li></ol>
<li>105 (25) uL 2X SYBR Fast</li></ol>
<li>Add 45 uL master mix to each sample</li>
<li>Add 45 uL master mix to each sample</li>
<li>Add 5 uL barcode oligo according to plate layout</li>
<li>Add 5 uL barcode oligo according to plate layout</li>


[[Image:|600px]]
[[Image:20170505-PlateLayout-BiotinOligoProduction.png|600px]]


<li>qPCR Cycles</li>
<li>qPCR Cycles</li>
Line 32: Line 32:
<li>16C hold</li>
<li>16C hold</li>
</ol></ol>
</ol></ol>
<li>Melting</li>
<li>Qiaquick Column</li>
<ol type="A">
<ol type="A">
<li>Withdraw 2XSSC buffer from end of protocol [[Daniel:Notebook/PosSequencing/2017-5-3|Yesterday]]</li>
<li>Add 250 PB (5X binding buffer) to 50 sample</li>
<li>Resuspend in 100μL of TE-Triton 0.1% (TE-T)</li>
<li>Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through</li>
<li>Melt off the complementary DNA strand by incubating the sample at 95°C for 3 minutes</li>
<li>Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through</li>
<li>Magnet pull-down for 1 minute and remove supernatant</li>
<li>Dry spin column for 1 minute at 14000 rpm; discard flow through</li>
<li>Add 100 μL of TE-T and mix</li>
<li>Let stand with cover open in fume hood for ~5 minutes</li>
<li>Repeat wash</li>
<li>Transfer column to a new 1.5 mL eppendorf tube</li>
<li>Add 35 uL nfH<sub>2</sub>O to column</li>
<li>Let stand 1 minute</li>
<li>Spin for 1 minute at 14000 rpm</li>
</ol>
</ol>
<li>Fluorescent Oligo Binding (All samples)</li>
<li>TBE Gel</li>
<ol type="A">
<ol type="A">
<li>Resuspend beads in 25 uL 4X SSC buffer</li>
<li>Mix 48 uL TBE and 12 uL 6X dye</li>
<li>Make the following dye hybridization buffer</li>
<li>Aliquot 10 uL mix to parafilm</li>
<ol type="a">
<li>Add 2 uL sample or 1.5 uL ladder to appropriate aliquot</li>
<li>8.4 each uL 10 uM dye; dcprobe6-488, dcprobe6-Cy3, dcprobe6-Cy5</li>
<li>Add 10 uL mix to appropriate lane</li>
<li>63 uL formamide</li>
<li>Run gel at 240V for 24 minutes</li>
<li>16.8 uL nfH<sub>2</sub>O</li></ol>
<li>Open gel and stain with 2 uL SYBR gold for 3 minutes</li>
<li>Add 25 uL fluorescent probe in 60% formamide to each sample</li>
<li>Rinse and image in gel doc</li>
<li>Incubate at room temp in the dark for 30 min</li>
<li>Wash twice in 2X SSC</li>
<li>Resuspend in 40 uL 2XSSC or 20 uL 2X SSC (Sample 1)</li>
</ol>
</ol>
</ol>
<li>Qubit</li>
<ol type="A">
<li>Mix 1194 uL dsDNA buffer and 6 uL dye</li>
<li>Aliquot 190 uL to standard tubes and 199 uL to dye tubes</li>
<li>Add 10 uL standard or 1 uL dye</li>
<li>Vortex and spin down</li>
<li>Let sit 5 minutes in the dark</li>
<li>Measure in the Qubit</li>
</ol></ol>
===Results===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#66FF66;font-size:12pt;font-weight:bold" align="center"
| width="160" height="37" | Sample
| width="65" | Qubit Conc (ug/mL)
| width="65" | Amount (nM)
| width="101" | Total Amount (fmol)
| width="81" | Amplification (Over 5 amol)
|- style="font-size:12pt"
| height="15"  valign="bottom" | Barcode Oligo v1
| align="center" align="center" valign="bottom" | 7.56
| align="center" align="center" valign="bottom" | 238.6
| align="center" align="center" valign="bottom" | 8352.3
| align="center" align="center" valign="bottom" | 1670460
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Barcode Oligo v2
| align="center" align="center" valign="bottom" | 4.85
| align="center" align="center" valign="bottom" | 153.1
| align="center" align="center" valign="bottom" | 5358.3
| align="center" align="center" valign="bottom" | 1071660
|- style="font-size:12pt"
| height="15"  valign="bottom" | Barcode Oligo v3
| align="center" align="center" valign="bottom" | 6.94
| align="center" align="center" valign="bottom" | 219.1
| align="center" align="center" valign="bottom" | 7667.3
| align="center" align="center" valign="bottom" | 1533460
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | NTC
| align="center" align="center" valign="bottom" | 0.12
| align="center" align="center" valign="bottom" | 3.8
| align="center" align="center" valign="bottom" | 132.6
| align="center" align="center" valign="bottom" | 26520
|}
<gallery perrow=2 heights=300px widths=300px</li>
File:20170505-qPCR-BarcodeOligoProduction.png|qPCR curves</li>
File:2017-05-05-BarcodeOligoProduction.png|Gel image
</gallery>
[[Category:PosSeq]] [[Category:20170505]] [[Category:OligoPrep]]

Latest revision as of 18:49, 22 May 2017

Probe Production[edit]

Back to Calendar

Dr Zhang had a good suggestion to do biotin probe production for each of the barcode oligos. I'll do this as a standard reaction without the bead oligos. I can then do the biotin attachment and oligo melting after that. This will allow me to separate the imaging steps from the actual emulsion PCR which will be more important for

Protocol[edit]

  1. qPCR
    1. Make 4.2X (1X) master mix with the following reagents
      1. 4.2 (1) uL 10 uM AP1v4U
      2. 4.2 (1) uL 10 uM BiotinU
      3. 75.6 (18) uL nfH2O
      4. 105 (25) uL 2X SYBR Fast
    2. Add 45 uL master mix to each sample
    3. Add 5 uL barcode oligo according to plate layout
    4. File:20170505-PlateLayout-BiotinOligoProduction.png
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold
  2. Qiaquick Column
    1. Add 250 PB (5X binding buffer) to 50 sample
    2. Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
    3. Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
    4. Dry spin column for 1 minute at 14000 rpm; discard flow through
    5. Let stand with cover open in fume hood for ~5 minutes
    6. Transfer column to a new 1.5 mL eppendorf tube
    7. Add 35 uL nfH2O to column
    8. Let stand 1 minute
    9. Spin for 1 minute at 14000 rpm
  3. TBE Gel
    1. Mix 48 uL TBE and 12 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 5 amol)
Barcode Oligo v1 7.56 238.6 8352.3 1670460
Barcode Oligo v2 4.85 153.1 5358.3 1071660
Barcode Oligo v3 6.94 219.1 7667.3 1533460
NTC 0.12 3.8 132.6 26520