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 | <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"> | ||
Line 22: | Line 22: | ||
<li>Mix together the three suspensions of beads with different oligos</li> | <li>Mix together the three suspensions of beads with different oligos</li> | ||
<li>Mix ingredients using the following table</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="340" height="45" | Component | |||
| width="85" | Ion Torrent Concentration | |||
| width="110" | My uL Added- Sample (Single) | |||
| width="180" | My Final Concentration | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | DreamTaq 10X PCR Buffer (20 mM MgCl2) | |||
| align="center" valign="bottom" | 1X | |||
| align="center" 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="center" valign="bottom" | 2.5 mM | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" valign="bottom" | 2.5 mM | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | 25 mM MgCl2 | |||
| align="center" valign="bottom" | 3.5 mM | |||
| align="center" align="center" valign="bottom" | 6 | |||
| align="center" valign="bottom" | 3.5 mM | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 100 uM Primer A (Free primer-Primer 2) | |||
| align="center" valign="bottom" | 9 uM | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" valign="bottom" | 5 uM | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | 10 uM Primer B (Bound primer-Primer 1) | |||
| align="center" valign="bottom" | 0.1 uM | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" valign="bottom" | 0.1 uM | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Hydrogel beads, 2M/uL | |||
| align="center" valign="bottom" | 50 Million | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" valign="bottom" | 60 M | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | DreamTaq Polymerase (5 U/uL) * | |||
| align="center" valign="bottom" | 90 Units | |||
| align="center" align="center" valign="bottom" | 18 | |||
| align="center" valign="bottom" | 90 Units | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="30" | DNA template 2nd Dilution (Barcode Oligos 1 pM) | |||
| align="center" | 0.12 pM | |||
| align="center" align="center" | 10 | |||
| align="center" valign="bottom" | 0.012 fmol total (0.006 fmol each?) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | 1 M Ammonium Sulfate | |||
| align="center" valign="bottom" | 5 mM | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 1 mM Spermidine*** | |||
| align="center" valign="bottom" | 100 uM | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | TIPP | |||
| align="center" valign="bottom" | NA | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Water | |||
| align="center" valign="bottom" | NA | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" valign="bottom" | NA | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Total Volume | |||
| align="center" align="center" valign="bottom" | | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 100 | |||
| align="center" align="center" valign="bottom" | | |||
|} | |||
<li>Aliquot 90 uL master mix into 4 tubes</li> | <li>Aliquot 90 uL master mix into 4 tubes</li> | ||
Line 50: | Line 137: | ||
<li>4C forever</li> | <li>4C forever</li> | ||
</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]])= | |||
==Protocol== | |||
<ol start="2"> | |||
<li>Qiaquick Column</li> | |||
'''Note''': I will be pooling samples and double loading columns, yielding 4 samples per column (2 columns per barcode oligo, 12 columns total) | |||
<ol type="A"> | |||
<li>Pool 4 samples and 1000 uL PB (5X binding buffer)</li> | |||
<li>Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through</li> | |||
<li>Add another 600 uL pooled sample to columnand spin for 1 minute at 14000 rpm; discard flow through | |||
<li>Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through</li> | |||
<li>Dry spin column for 1 minute at 14000 rpm; discard flow through</li> | |||
<li>Let stand with cover open in fume hood for ~5 minutes</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> | |||
<li>Pool samples when finished; concentrate if desired</li> | |||
</ol> | |||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 160 uL TBE and 40 uL 6X dye</li> | |||
<li>Aliquot 10 uL mix to parafilm</li> | |||
<li>Add 2 uL sample or 1.5 uL ladder to appropriate aliquot</li> | |||
<li>Add 10 uL mix to appropriate lane</li> | |||
<li>Run gel at 240V for 24 minutes</li> | |||
<li>Open gel and stain with 2 uL SYBR gold for 3 minutes</li> | |||
<li>Rinse and image in gel doc</li> | |||
</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="190" height="45" | Sample | |||
| width="85" | Qubit Conc (ug/mL) | |||
| width="100" | Amount (nM) | |||
| width="95" | Total Amount (fmol) | |||
| width="95" | Amplification (Over 40 amol) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Barcode Oligo v1 (EcoRI) | |||
| align="center" align="center" | 33.0 | |||
| align="center" align="center" | 526.3 | |||
| align="center" align="center" | 3.16E+04 | |||
| align="center" align="center" | 7.89E+08 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Barcode Oligo v2 (EcoRI) | |||
| align="center" align="center" | 20.0 | |||
| align="center" align="center" | 319.0 | |||
| align="center" align="center" | 2.87E+04 | |||
| align="center" align="center" | 7.18E+08 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Barcode Oligo v3 (EcoRI) | |||
| align="center" align="center" | 33.7 | |||
| align="center" align="center" | 537.5 | |||
| align="center" align="center" | 3.22E+04 | |||
| 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> | |||
File:2017-05-25-PosSeq-BarcProduction-EcoRI.png|EcoRI Samples | |||
File:2017-05-25-PosSeq-BarcProduction-Uracil.png|Uracil samples | |||
</gallery> | |||
[[Category:20170524]] [[Category:20170525]] [[Category:OligoPrep]] [[Category:PosSeq]] |
Latest revision as of 19:55, 31 May 2017
Emulsion PCR Test 2[edit]
From the results on
Protocol[edit]
- Oil Production
- Start from Monday April 10's oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)
- Template-Bead Binding; Make 4 samples and follow this protocol for each
- Suspend 5 uL (50 ug, ~50M beads) beads in 100 uL Dynabuffer
- Apply magnet for 30 sec and remove supernatant
- Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)
- 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
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Repeat wash step
- Resuspend beads in 10 uL TE buffer
- Mix together the three suspensions of beads with different oligos
- Mix ingredients using the following table
- Aliquot 90 uL master mix into 4 tubes
- Add 10 uL appropriate primers according to sample table
- Aliquot 50 uL each non-emulsion sample into new tubes (A/B)
- Oil-Aqueous Mixing
- Mix 20 uL aqueous into 200 uL oil mixture
- Vortex for 30 seconds
- Repeat mixing to 100 uL (5 times)
- Mix with vortexer for 3 minutes
- Dilute with pure mineral oil for imaging (1:100)
- Distribute 300 uL total volume into 6 tubes, 50 uL each; use same pipette tip; only made 5 (oil sticks to tip)
- Emulsion PCR
- Perform PCR with the following settings
- 95C 2 min
- 95C 20 sec
- 58C 30 sec
- 70C 30 sec
- Goto b 40 times
- 95C 30 sec
- 68C 3 min
- Goto f 20X times
- 4C forever
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 |
Continued Tuesday, May 30
Barcode Oligo Production (Started Yesterday)[edit]
Protocol[edit]
- Qiaquick Column Note: I will be pooling samples and double loading columns, yielding 4 samples per column (2 columns per barcode oligo, 12 columns total)
- Pool 4 samples and 1000 uL PB (5X binding buffer)
- Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
- Add another 600 uL pooled sample to columnand spin for 1 minute at 14000 rpm; discard flow through
- Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
- Dry spin column for 1 minute at 14000 rpm; discard flow through
- Let stand with cover open in fume hood for ~5 minutes
- Transfer column to a new 1.5 mL eppendorf tube
- Add 35 uL nfH2O to column
- Let stand 1 minute
- Spin for 1 minute at 14000 rpm
- Pool samples when finished; concentrate if desired
- TBE Gel
- Mix 160 uL TBE and 40 uL 6X dye
- Aliquot 10 uL mix to parafilm
- Add 2 uL sample or 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to appropriate lane
- Run gel at 240V for 24 minutes
- Open gel and stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
- Qubit
- Mix 1194 uL dsDNA buffer and 6 uL dye
- Aliquot 190 uL to standard tubes and 199 uL to dye tubes
- Add 10 uL standard or 1 uL dye
- Vortex and spin down
- Let sit 5 minutes in the dark
- 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 |
- 2017-05-25-PosSeq-BarcProduction-EcoRI.png
EcoRI Samples
- 2017-05-25-PosSeq-BarcProduction-Uracil.png
Uracil samples