Daniel:Notebook/PosSequencing/2017-7-12: Difference between revisions
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=emPCR 9-Concentration Comparison= | =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. | |||
[[Category:PosSeq]] [[Category:20170706]] | ==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="280" height="45" | Component | |||
| width="95" | uL Added | |||
| width="100" | 3.2X Master Mix uL Added | |||
| width="120" | 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" | 0 | |||
| 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" | 2 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" valign="bottom" | 10 Units | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" | Barcode Oligo v1 or v3 (template, 100 pM) | |||
| align="center" align="center" | 5 | |||
| align="center" align="center" | 0 | |||
| align="center" | 10 pM | |||
|- 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" | TIPP | |||
| align="center" align="center" valign="bottom" | 0 | |||
| 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" align="center" valign="bottom" | 26.5 | |||
| align="center" align="center" valign="bottom" | 84.8 | |||
| align="center" valign="bottom" | NA | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Total Volume | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 95 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 249.6 | |||
| align="center" align="center" valign="bottom" | | |||
|} | |||
<li>Add 83 uL of master mix to each set of 10 uL beads</li> | |||
<li>Add barcode oligos and water mix according to table</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#9BBB59;font-size:12pt;font-weight:bold" | |||
| align="right" width="240" height="28" valign="bottom" | | |||
| width="95" align="center" | Sample 1 | |||
| width="95" align="center" | Sample 2 | |||
| width="95" align="center" | Sample 3 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Barcode Oligo v1 (100 pM) | |||
| align="right" align="center" valign="bottom" | 5 | |||
| align="right" align="center" valign="bottom" | 0 | |||
| align="right" align="center" valign="bottom" | 5 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Barcode Oligo v3 (100 pM) | |||
| align="right" align="center" valign="bottom" | 0 | |||
| align="right" align="center" valign="bottom" | 5 | |||
| align="right" align="center" valign="bottom" | 5 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="right" align="center" valign="bottom" | 5 | |||
| align="right" align="center" valign="bottom" | 5 | |||
| align="right" align="center" valign="bottom" | 0 | |||
|} | |||
<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]] |
Revision as of 22:38, 12 July 2017
emPCR8 (Started Thursday July 6)
Protocol (From Yesterday)
- Dye hybridization
- Mix 20 uL ssDNA (lambda digested) and 20 uL 4X SSC buffer
- Incubate in the dark at 70C for 5 minutes
- Incubate at 55C for 20 minutes
- Add 1 uL appropriate (10 uM) dye, A488-dc6 for sample 1 and Cy5-dc6 for sample 3
- Incubate in the dark (RT) for 15 minutes
- Magnet pulldown, remove supernatant and add 40 uL 2X SSC buffer
- Add 15 uL to a slide for analysis
- Seal coverslip with nail polish and wait approximately 30 minutes to dry
Results
- 20170712-emPCR8 Barcode3-Field4-SNAP ch00.jpg
Field 4
- 20170712-emPCR8 Barcode3-Field5-SNAP ch00.jpg
Field 5
- 20170712-emPCR8 Barcode3-Field6-SNAP ch00.jpg
Field 6
- 20170712-emPCR8 Barcode-v1v3-Field1-SNAP ch00.jpg
Field 1
- 20170712-emPCR8 Barcode-v1v3-Field2-SNAP ch00.jpg
Field 2
- 20170712-emPCR8 Barcode-v1v3-Field5-SNAP ch00.jpg
Field 5
- 20170712-emPCR8 Barcode-v1v3-Field6-SNAP ch00.jpg
Field 6
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
- 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 3 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
- Add 83 uL of master mix to each set of 10 uL beads
- Add barcode oligos and water mix according to table
- Add 2 uL DreamTaq Polymerase to each sample
- 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
- 45C 30 sec
- 70C 30 sec
- Goto b 95 times
- 4C forever
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 | 0 | 25 M |
DreamTaq Polymerase (5 U/uL) * | 2 | 0 | 10 Units |
Barcode Oligo v1 or v3 (template, 100 pM) | 5 | 0 | 10 pM |
100 mM Ammonium Sulfate | 5 | 16 | 5 mM |
1 mM Spermidine*** | 10 | 32 | 100 uM |
TIPP | 0 | 0 | 0 |
Water | 26.5 | 84.8 | NA |
Total Volume | 95 | 249.6 |
Sample 1 | Sample 2 | Sample 3 | |
Barcode Oligo v1 (100 pM) | 5 | 0 | 5 |
Barcode Oligo v3 (100 pM) | 0 | 5 | 5 |
nfH2O | 5 | 5 | 0 |
******
Continued tomorrow