Daniel:Notebook/PosSequencing/2017-5-31: Difference between revisions
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[[Category:PosSeq]] [[Category:20170525]] | [[Category:PosSeq]] [[Category:20170525]] [[Category:20170531]] |
Revision as of 19:57, 31 May 2017
Emulsion PCR Test 2 (Started Thursday May 25)
Yesterday's imaging showed no results. As a quick check I'm going to use SYBR to check the presence of any DNA product. I'll use a positive and negative control to analyze the results more effectively.
Protocol
- SYBR Check
- Centrifuge sample at 13,000rpm for 1 minute
- Remove supernatant except about 10 uL
- Add 10 uL SYBR Green; incubate room temp 5 min
- Centrifuge sample at 13,000rpm for 1 minute
- Remove supernatant except about 10 uL
- Add ~40 uL 2X SSC buffer; total volume should be about 50 uL
- Add 15 uL to slide for analysis
- Empty Beads SYBR Loading (Negative Control)
- Add 2 uL beads (20 ug, 20 million beads) to 100 uL wash buffer
- Pull down with magnet for 30 seconds and remove supernatant
- Add 10 uL nfH2O and 10 uL 2X SYBR mix; incubate at RT for 5 minutes
- Pull down with magnet for 30 seconds and remove supernatant
- Add 50 uL 2X SSC buffer
- Add 15 uL to slide for analysis
- dsDNA SYBR Loading (Positive Control)
- Add 2 uL beads (20 ug, 20 million beads) to 100 uL wash buffer
- Pull down with magnet for 30 seconds and remove supernatant
- Add 20 uL Dynabuffer, 15 uL nfH2O, and 5 uL 500 nM barcode 2 oligo
- Incubate at RT for 15 minutes
- Pull down with magnet for 30 seconds and remove supernatant
- Wash with 100 uL dynabuffer
- Pull down with magnet for 30 seconds and remove supernatant
- Add 10 uL nfH2O and 10 uL 2X SYBR mix; incubate at RT for 5 minutes
- Pull down with magnet for 30 seconds and remove supernatant
- Add 50 uL 2X SSC buffer
- Add 15 uL to slide for analysis
- Oil phase
- Mix 5 uL Triton X, 40 uL Tween-80, and 450 uL Span 80 into 9.5 mL Mineral oil
- Vortex vigorously to mix
- 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
******
******
Emulsion PCR Test 3
This emulsion test will be aimed at determining the success of the actual emPCR using the qPCR checks. For that I'll be taking samples at different times. See the workflow below.
File:20170531-emPCR-Workflow.png
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 tomorrow