Daniel:Notebook/PosSequencing/2017-5-8
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Probe Production (Started Friday May 5)
Protocol
After the success on Friday, I want to redo the production protocol but for the EcoRI primer. This makes it so that I can mix the two in relative quantities and test out the USER cleavage protocol.
- qPCR
- Make 4.2X (1X) master mix with the following reagents
- 4.2 (1) uL 10 uM AP1v4U
- 4.2 (1) uL 10 uM BiotinEcoRI
- 75.6 (18) uL nfH2O
- 105 (25) uL 2X SYBR Fast
- Add 45 uL master mix to each sample
- Add 5 uL barcode oligo according to plate layout File:20170505-PlateLayout-BiotinOligoProduction.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- Qiaquick Column
- Add 250 PB (5X binding buffer) to 50 sample
- Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
- Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
- Dry spin column for 1 minute at 14000 rpm; discard flow through
- Let stand with cover open in fume hood for ~5 minutes
- Transfer column to a new 1.5 mL eppendorf tube
- Add 35 uL nfH2O to column
- Let stand 1 minute
- Spin for 1 minute at 14000 rpm
- TBE Gel
- Mix 48 uL TBE and 12 uL 6X dye
- Aliquot 10 uL mix to parafilm
- Add 2 uL sample or 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to appropriate lane
- Run gel at 240V for 24 minutes
- Open gel and stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
- Qubit
- Mix 1194 uL dsDNA buffer and 6 uL dye
- Aliquot 190 uL to standard tubes and 199 uL to dye tubes
- Add 10 uL standard or 1 uL dye
- Vortex and spin down
- Let sit 5 minutes in the dark
- Measure in the Qubit
Results
Sample | Qubit Conc (ug/mL) | Amount (nM) | Total Amount (fmol) | Amplification (Over 5 amol) |
Barcode Oligo v1 | 9.30 | |||
Barcode Oligo v2 | 11.9 | |||
Barcode Oligo v3 | 8.01 | |||
NTC | 0.15 |
Bead Binding Test
- Bead Binding
- Suspend 2 uL (20 ug, ~20M 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 template oligo and up to 20 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
- Melting
- Withdraw 2XSSC buffer from end of protocol Yesterday
- Resuspend in 100μL of TE-Triton 0.1% (TE-T)
- Melt off the complementary DNA strand by incubating the sample at 95°C for 3 minutes
- Magnet pull-down for 1 minute and remove supernatant
- Add 100 μL of TE-T and mix
- Repeat wash
- Fluorescent Oligo Binding (All samples)
- Resuspend beads in 25 uL 4X SSC buffer
- Make the following dye hybridization buffer
- 8.4 each uL 10 uM dye; dcprobe6-488, dcprobe6-Cy3, dcprobe6-Cy5
- 63 uL formamide
- 16.8 uL nfH2O
- Add 25 uL fluorescent probe in 60% formamide to each sample
- Incubate at room temp in the dark for 30 min
- Wash twice in 2X SSC
- Resuspend in 40 uL 2XSSC or 20 uL 2X SSC (Sample 1)