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 m (→Protocol) |
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(7 intermediate revisions by the same user not shown) | |||
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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" | | |||
|} | |||
</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]
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]
- Strand Separation (DNA Denaturation)
- Add 500 uL KOH buffer (pH 12.0)
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL buffer
- Repeat KOH steps A-C once more
- Add 500 uL 4X SSC buffer (pH 12.0)
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 10uL buffer
- Resuspend in 80 uL 2X SSC buffer
- Single primer qPCR
- Make the following master mixes
- Aliquot 35 uL master mix to appropriate lanes
- Add 2 uL sample according to plate layout below File:PlateLayout-Beadless-emPCR-singleprimer-20170626.png
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 45C 30 sec
- 72C 20 sec
- plate read
- goto b x90
- 72C 2 min
- 16C hold
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 |
Results[edit]
- PlateLayout-Beadless-emPCR-singleprimer-20170626.png
Plate layout
- Beadless-emPCR-singleprimer-20170626-plateCTs-normed.png
Plate CTs
- Beadless-emPCR-singleprimer-20170626-rawcurves.png
Raw curves
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]
- Oil phase
- Mix 5 uL Triton X, 40 uL Tween-80, and 450 uL Span 80 into 2 mL Mineral oil and 8 mL Tegosoft DEC
- Vortex vigorously to mix
- Template-Bead Binding; Make 4 samples and follow this protocol for each
- Suspend 2.5 uL (25 ug, ~25M 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 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
- Aqueous Phase Preparation
- 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 ingredients using the following table
- Oil-Aqueous Mixing
- Mix 10 uL aqueous into 100 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 150 uL total volume into 3 tubes, 50 uL each; use same pipette tip
- Emulsion PCR
- Perform PCR with the following settings
- 95C 2 min
- 95C 20 sec
- 58C 30 sec
- 70C 30 sec
- Goto b 95 times
- 4C forever
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 |
Continued tomorrow