Daniel:Notebook/PosSequencing/2017-5-17
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Bead Binding Test 3
Trying to test the bead binding and denaturation on the beads again. This time we'll use high pH as the denaturant. From the paper above it says the streptavidin bond is stable to about pH 11, and according to this website DNA denaturation occurs at pH>10. I will use the alkaline lysis solution from Andrew's notes
Alkaline Lysis Solution
- 97 ul Nuclease free H2O.
- 1 ul 100 mM KOH
- 2 ul 0.5 M EDTA
Neutralization Solution
- 3 mL H20
- 3 mL 2M Tris-HCl
- 4 mL 1N HCl
Protocol
Sample Matrix
Sample # | Condition |
1 | Barcode Oligo 2 |
2 | Barcode Oligo 2-No Melting |
- Bead Binding
- Suspend 1 uL (10 ug, ~5M beads designed at 2.5 pmol) beads in 100 uL Dynabuffer
- Apply magnet for 30 sec and remove supernatant
- Suspend in 10 uL Dynabuffer (conc. 1 ug/uL)
- Add 5 uL barcode oligo and up to 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
- Add 100 uL wash buffer and 1 uL DAPI; incubate in the dark for 5 minutes;
- Add 15 uL to a slide for analysis
- Remove the rest of the supernatant after bead pulldown
- Melting
- 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; Magnet pulldown and remove supernatant
- Add 100 μL of TE-T and mix
- Add 15 uL to a slide for analysis
- Remove the rest of the supernatant after bead pulldown
- Hold here and wait for results from slides
- Fluorescent Oligo Binding (All samples)
- Resuspend beads in 25 uL 4X SSC buffer
- Make the following 3.1X (1X) dye hybridization buffer
- 6.2 (2 ea; 6 total) each uL 10 uM dye; dcprobe6-488, dcprobe6-Cy3, dcprobe6-Cy5
- 46.5 (15) uL formamide
- 12.4 (4) uL nfH2O
- Incubate probes at 80C for 5 minutes
- 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