Daniel:Notebook/PosSequencing/2017-6-22: Difference between revisions
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|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | 10 uM dual biotin primer | | height="15" valign="bottom" | 10 uM dual biotin primer | ||
| align="right" align="center" valign="bottom" | | | align="right" align="center" valign="bottom" | 2 | ||
| align="right" align="center" valign="bottom" | | | align="right" align="center" valign="bottom" | 2 | ||
| align="center" valign="bottom" | 0. | | align="center" valign="bottom" | 0.2 uM * | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
Line 95: | Line 95: | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Water | | height="15" valign="bottom" | Water | ||
| align="right" align="center" valign="bottom" | | | align="right" align="center" valign="bottom" | 45 | ||
| align="right" align="center" valign="bottom" | | | align="right" align="center" valign="bottom" | 45 | ||
| align="center" valign="bottom" | NA | | align="center" valign="bottom" | NA | ||
Line 106: | Line 106: | ||
|} | |} | ||
* Note: This is based off normally adding in 10 uL of beads containing 12.5 pmol biotinylated primer + 0.5 uL 10 uM primer = 17.5 pmol =0.175uM final concentration. The increased aqueous addition makes the final concentration 0.2uM | |||
</ol> | </ol> | ||
Line 123: | Line 125: | ||
<li>95C 2 min</li> | <li>95C 2 min</li> | ||
<li>95C 20 sec</li> | <li>95C 20 sec</li> | ||
<li> | <li>45C 30 sec</li> | ||
<li>70C 30 sec</li> | <li>70C 30 sec</li> | ||
<li>Goto b 50 times</li> | <li>Goto b 50 times</li> | ||
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</ol> | </ol> | ||
Continued [[Daniel:Notebook/PosSequencing/2017-6-23|tomorrow]] | |||
[[Category:PosSeq]] [[Category:20170622]] | [[Category:PosSeq]] [[Category:20170622]] |
Latest revision as of 16:25, 23 June 2017
Beadless emPCR[edit]
This is to test the emPCR without using beads.
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
- 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
- Note: This is based off normally adding in 10 uL of beads containing 12.5 pmol biotinylated primer + 0.5 uL 10 uM primer = 17.5 pmol =0.175uM final concentration. The increased aqueous addition makes the final concentration 0.2uM
- 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 50 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 | 2 | 2 | 0.2 uM * |
Hydrogel beads, 2M/uL | 0 | 0 | 0 |
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 | 45 | 45 | NA |
Total Volume | 100 | 100 |
Continued tomorrow