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=Bead Binding Test 3-Alkaline Denaturation= [[Daniel:Notebook/PosSequencing|Back to Calendar]] [[Media:Kohler_Avidin-Streptavidin_Bonds_Molecules_2014.pdf|Avidin-Biotin Bonds Paper]] 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 [https://biology.stackexchange.com/questions/29925/why-does-high-ph-result-in-the-denaturation-of-dna this website] DNA denaturation occurs at pH>10. I will use the alkaline lysis solution from [[Arichard:Reagents/als|Andrew's notes]] '''Alkaline Lysis Solution''' *97 ul Nuclease free H<sub>2</sub>O. *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 ==Dye Test== Simple test to determine viability of the SYBR gree dye (from Kapa SYBR Master Mix) for the assay. <ol> <li>Bead Binding</li> <ol type="A"> <li>Suspend 1 uL (10 ug, ~5M beads designed at 2.5 pmol) beads in 100 uL Dynabuffer</li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Suspend in 10 uL Dynabuffer (conc. 1 ug/uL)</li> <li>Add 5 uL barcode oligo,either PCCT (10 uM, ssDNA) or Barcode Oligo 2, and up to 5 uL nfH<sub>2</sub>O per sample to bead solution; incubate at RT for 15 min</li> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> <li>Repeat wash step</li> <li>Add 50 uL Kapa SYBR Fast master mix 2X + nfH<sub>2</sub>O at a 1:1 ratio; incubate in the dark for 15 minutes;</li> <li>Bead pulldown and remove supernatant</li> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> <li>Resuspend in 50 uL Dynabeads buffer</li> <li>Add 15 uL to a slide for analysis</li> <li>Check in microscope</li> </ol> </ol> ==Protocol== '''Sample Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | width="100" height="39" | Sample # | width="160" | Condition | width="120" | uL 100mM KOH in 100 uL | width="120" | uL 1 mM KOH in 100 uL | width="85" | uL 0.5 M EDTA | width="85" | uL nfH<sub>2</sub>O |- style="font-size:12pt" align="center" | align="center" height="15" | 1 | pH 12 | align="center" | 10 | NA | align="center" | 2 | align="center" | 88 |- style="background-color:#BFBFBF;font-size:12pt" align="center" | align="center" height="15" | 2 | pH 11 | align="center" | 1 | NA | align="center" | 2 | align="center" | 97 |- style="font-size:12pt" align="center" | align="center" height="15" | 3 | pH 10 | NA | align="center" | 10 | align="center" | 2 | align="center" | 88 |- style="background-color:#BFBFBF;font-size:12pt" align="center" | align="center" height="15" | 4 | pH 9 | NA | align="center" | 1 | align="center" | 2 | align="center" | 97 |- style="font-size:12pt" align="center" | align="center" height="15" | 5 | No denaturation | align="center" | 0 | align="center" | 0 | align="center" | 2 | align="center" | 98 |} <ol> <li>Bead Binding</li> <ol type="A"> <li>Suspend 1 uL (10 ug, ~5M beads designed at 2.5 pmol) beads in 100 uL Dynabuffer</li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Suspend in 10 uL Dynabuffer (conc. 1 ug/uL)</li> <li>Add 5 uL barcode oligo and up to 5 uL nfH<sub>2</sub>O per sample to bead solution; incubate at RT for 15 min</li> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> <li>Add 100 uL wash buffer and 1 uL SYBR; incubate in the dark for 5 minutes;</li> <li>Add 15 uL to a slide for analysis</li> <li>Remove the rest of the supernatant after bead pulldown</li> </ol> <li>Melting</li> <ol type="A"> <li>Resuspend in 100μL of Alkaline Solution-See Sample Matrix above</li> <li>Wait 3 minutes</li> <li>Magnet pull-down for 1 minute and remove supernatant</li> <li>Add 100 μL of wash buffer and mix; Magnet pulldown and remove supernatant</li> <li>Add 100 μL of wash buffer and mix</li> <li>Add 15 uL to a slide for analysis</li> <li>Remove the rest of the supernatant after bead pulldown</li> <li>Hold here and wait for results from slides</li> </ol> </ol> [[Category:PosSeq]] [[Category:20170508]]
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