Daniel:Notebook/PosSequencing/2017-4-24: Difference between revisions
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[[Daniel:Notebook/PosSequencing|Back to Calendar]] | [[Daniel:Notebook/PosSequencing|Back to Calendar]] | ||
Not actually run today. Probably [[Daniel:Notebook/PosSequencing/2017-4-26|Wednesday]]. | |||
==Protocol== | ==Protocol== | ||
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<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> | <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> | ||
<li>Repeat wash step</li> | <li>Repeat wash step</li> | ||
<li>Resuspend beads in | <li>Resuspend beads in 5 uL TE buffer</li> | ||
<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>Prepare the aqueous mix according to the table below</li> | |||
<li> | |||
<li>Mix ingredients using the following table (see '''Notes''')</li> | <li>Mix ingredients using the following table (see '''Notes''')</li> | ||
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|} | |} | ||
</ol> | |||
<li>Oil-Aqueous Mixing</li> | |||
<ol type="A"> | |||
<li>Mix 20 uL aqueous into 200 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 300 uL total volume into 6 tubes, 50 uL each</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>68C 30 sec</li> | |||
<li>Goto b 40 times</li> | |||
<li>95C 30 sec</li> | |||
<li>68C 6 min</li> | |||
<li>Goto f 20X times</li> | |||
<li>4C forever</li> | |||
</ol></ol> | </ol></ol> | ||
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<li>Repeat steps 11 to 15 two times (j to o)</li> | <li>Repeat steps 11 to 15 two times (j to o)</li> | ||
<li>Resuspend in ~100 uL 4X SSC buffer</li></ol> | <li>Resuspend in ~100 uL 4X SSC buffer</li></ol> | ||
<li>Fluorescent | <li>Fluorescent Oligo Binding</li> | ||
<li></li> | |||
<li>Imaging</li> | <li>Imaging</li> | ||
</ol> | </ol> | ||
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==Notes== | ==Notes== | ||
* | * *: NEB Taq has terrible information. Since the ThermoFisher Taq is much better documented and available at a reasonable price, I will go with that in the future. It is on the order sheet and I will switch to that in the future. Since I'm not running the protocol today (It's almost 5), the Thermo | ||
* | * **: dNTPs are also available at up to 100 mM for each dNTP if this concentration proves too annoying to work with</li> | ||
* | * ***: Spermidine seems useful so I'm ordering it. We may actually have Ammonium sulfate but I'm not sure it's necessary</li> |
Latest revision as of 00:30, 25 April 2017
Emulsion PCR[edit]
Not actually run today. Probably Wednesday.
Protocol[edit]
This protocol is principally based off the Ion Torrent protocol for emulsion PCR
- Template DNA Production (Barcode Oligos)
- Start with oligo concentrations of 100 uM (100 pmol/uL)
- Make dilutions by adding 5 uL into 50 uL (1:10 dilutions)
- Dilute 8X times (final concentration 1 pM or 1 amol/uL)
- Make oligo solution by adding 5 uL barcode1 (Alexa488) and barcode3(Cy5) since they have low overlap-Final concentration of oligo is 1 pM; adding 10 uL to PCR mix makes it 0.1pM, the suggested concentration from Ion Torrent protocol
- Oil Production
- Start from Monday April 14's oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)
- Template-Bead Binding
- Suspend 2.5 uL (25 ug) beads in 100 uL Dynabuffer
- Apply magnet for 30 sec and remove supernatant
- Suspend 2.5 uL (25 ug) beads in 20 uL Dynabuffer (conc. 1 ug/uL)
- Add 10 uL 1 uM (10 pmol total, 10X excess) template oligo and 10 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 5 uL TE buffer
- Mix together the three suspensions of beads with different oligos
- Prepare the aqueous mix according to the table below
- Mix ingredients using the following table (see Notes)
- 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
- Emulsion PCR
- Perform PCR with the following settings
- 95C 2 min
- 95C 20 sec
- 58C 30 sec
- 68C 30 sec
- Goto b 40 times
- 95C 30 sec
- 68C 6 min
- Goto f 20X times
- 4C forever
- Emulsion Breaking
- With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside
- Add 100 uL isopropyl alcohol (IPA) to tube with 200 uL tip (use new tip)
- Draw up IPA with tip from part A, mix to resuspend tip
- Vortex the pooled sample in the 1.5 mL tube for 30 seconds
- Spin down the sample 13,000 rpm for 5 minutes
- Take off oil-IPA mix (top layer) but do not disrupt the oil/aqueous interface
- Add 1000 uL IPA and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Again remove IPA without disturbing the pellet
- Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds
- Go back and mix thoroughly by pipetting up and down; the pellet may be hard to resuspend
- Leave on 1.5mL mixer at 1300rpm 1 minute
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL wash buffer
- Repeat steps 11 to 15 two times (j to o)
- Resuspend in ~100 uL 4X SSC buffer
- Fluorescent Oligo Binding
- Imaging
- *: NEB Taq has terrible information. Since the ThermoFisher Taq is much better documented and available at a reasonable price, I will go with that in the future. It is on the order sheet and I will switch to that in the future. Since I'm not running the protocol today (It's almost 5), the Thermo
- **: dNTPs are also available at up to 100 mM for each dNTP if this concentration proves too annoying to work with
- ***: Spermidine seems useful so I'm ordering it. We may actually have Ammonium sulfate but I'm not sure it's necessary
Component | Ion Torrent Concentration | My uL Added | My Final Concentration |
5X PCR Buffer (Taq) * | 1X | 20 | 1X |
10 mM dNTPs ** | 2.5 mM | 25 | 2.5 mM |
25 mM MgCl2 | 3.5 mM | 14 | 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 |
Taq? Polymerase (5 U/uL) * | 90 Units | 10 | |
DNA template 2nd Dilution (Barcode Oligos) | 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 | 0 | 0 |
TIPP | NA | 0 | 0 |
Water | NA | 5 | NA |
Total Volume | 100 |