Daniel:Notebook/PosSequencing/2017-6-6
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Aqueous PCR 2
Last time I may have been using too much enzyme. Too high of glycerol can inhibit PCR, and
Protocol
- Template-Bead Binding
- Suspend 12 uL (120 ug, ~120M 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 2.5 uL 100 uM Biotin-Uracil primer1 (250 pmol total, 2X excess) and 17.5 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 30 uL TE buffer
- qPCR
- Mix together the three suspensions of beads with different oligos
- Mix ingredients using the following table
- Add 33.5 uL master mix to each lane
- Add Template, Taq, and nfH2O as relevant and dictated by table/plate layout File:PlateLayout-20170606-aqPCR2 .png
- 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
Component | IT Concentration | Sample (Single) | Sample Concentration | No SYBR | Overloaded | 10 Units | NTC | 5.2X Master Mix | Master Mix Applicable |
DreamTaq 10X PCR Buffer (20 mM MgCl2) | 1X | 5 | 1X | 5 | 5 | 5 | 5 | 26 | 1 |
10 mM dNTPs | 2.5 mM | 12.5 | 2.5 mM | 12.5 | 12.5 | 12.5 | 12.5 | 65 | 1 |
25 mM MgCl2 | 3.5 mM | 3 | 3.5 mM | 3 | 3 | 3 | 3 | 15.6 | 1 |
100 uM Primer A (Free primer-Primer 2) | 9 uM | 2.5 | 5 uM | 2.5 | 2.5 | 2.5 | 2.5 | 13 | 1 |
10 uM Primer B (Bound primer-Primer Biotin-Uracil) | 0.1 uM | 0.5 | 0.1 uM | 0.5 | 0.5 | 0.5 | 0.5 | 2.6 | 1 |
Hydrogel beads, 2M/uL | 50 Million | 5 | 60 M | 5 | 5 | 5 | 5 | 26 | 1 |
DreamTaq Polymerase (5 U/uL) * | 90 Units/150 uL | 6 | 30 Units | 6 | 9 | 2 | 6 | 0 | 0 |
DNA template 2nd Dilution (Barcode Oligo v2 1 pM) | 0.12 pM | 5 | 0.01 fmol | 5 | 5 | 5 | 0 | 0 | 0 |
1 M Ammonium Sulfate | 5 mM | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
1 mM Spermidine*** | 100 uM | 5 | 0 | 5 | 5 | 5 | 5 | 26 | 1 |
TIPP | NA | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
Water | NA | 4.5 | NA | 5.5 | 1.5 | 8.5 | 9.5 | 0 | 0 |
SYBR Green (100:1) | NA | 1 | NA | 0 | 1 | 1 | 1 | 0 | 0 |
Total Volume | 50 | 50 | 50 | 50 | 50 | 174.2 |