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==Protocol-Part 2== <ol start="6"> <li>Emulsion Breaking</li> <ol type="A"> <li>With a 200 uL pipette add 100 uL diethyl ether to each sample; save the tip</li> <li>Adjust tip to 200 and using same tip pool samples in a new 1.5 mL tube</li> <li>Vortex the pooled sample in the 1.5 mL tube for 30 seconds</li> <li>Spin down the sample 13,000 rpm for 5 minutes</li> <li>Take off oil-DE mix (top layer) but do not disrupt the oil/aqueous interface</li> <li>Add 1000 uL ethyl acetate and vortex for 30 seconds</li> <li>Spin down sample at 13,000rpm for 3 minutes</li> <li>Remove ethyl acetate without disturbing the pellet</li> <li>Add 1000 uL diethyl ether and vortex for 30 seconds</li> <li>Spin down sample at 13,000rpm for 3 minutes</li> <li>Remove DE without disturbing the aqueous phase</li> <li>Allow DE to dry in the fume hood; <span style="background:#FFFF00">resuspend beads in the solution and take 15 uL aside for testing</span></li> </ol> <li>Phosphorylation</li> <ol type="A"> <li>Pull down beads, remove supernatant, and wash with 100 uL DynaT-buffer (Dynabuffer + 0.05% Tween20)</li> <li>Pull down beads and remove supernatant</li> <li>Resuspend the beads in the following mixture</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | width="200" height="42" | Reagent | width="95" | uL 1X Reaction | width="95" | uL 3.2X Master Mix |- style="font-size:12pt" | height="15" valign="bottom" | T4 PNK Buffer (10X) | align="right" align="center" valign="bottom" | 5 | align="right" align="center" valign="bottom" | 16 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | ATP (10 mM) | align="right" align="center" valign="bottom" | 5 | align="right" align="center" valign="bottom" | 16 |- style="font-size:12pt" | height="15" valign="bottom" | T4 PNK (10 U/uL) | align="right" align="center" valign="bottom" | 1 | align="right" align="center" valign="bottom" | 3.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="right" align="center" valign="bottom" | 39 | align="right" align="center" valign="bottom" | 124.8 |- style="font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="right" align="center" valign="bottom" | 50 |style="font-weight:bold" align="right" align="center" valign="bottom" | 160 |} <li>Resuspend each sample in 49 uL master mix</li> <li>Add 1 uL T4 PNK to each</li> <li>Incubate for 30 minutes at 37C</li> <li>Heat inactivate by incubating for 20 minutes at 65C</li> </ol> <li>Lambda Exonuclease Digestion</li> <ol type="A"> <li>Pull down with magnet and remove supernatant</li> <li>Make the following master mix</li> <ol type="a"><li>16 uL 10X Lambda exonuclease reaction buffer</li><li>140.8 uL nfH<sub>2</sub>O</li></ol> <li>Resuspend beads in 49 uL master mix</li> <li>Add 1 uL lambda exonuclease (10 U/uL)</li> <li>Incubate for 30 minutes at 37C</li> <li>Heat kill enzyme by incubating for 10 minutes at 80C</li> <li>Magnet pulldown, and resuspend in 50 uL 2XSSC</li> </ol> <li>Dye hybridization</li> <ol type="A"> <li>Aliquot 20 uL sample to a new tube</li> <li>Add 20 uL 4X SSC and 50% formamide</li> <li>Add 1 uL appropriate (10 uM) dye, A488-dc6 for sample 1 and Cy5-dc6 for sample 3</li> <li>Incubate in the dark (RT) for 15 minutes</li> <li>Magnet pulldown, remove supernatant and add 40 uL 2X SSC buffer</li> <li>Add 15 uL to a slide for analysis</li> <li>Seal coverslip with nail polish and wait approximately 30 minutes to dry</li> </ol> <li>Imaging Parameters</li> <ol type="A"> <li>Confocal Microscope</li> <li>488 Laser-3.00%</li> <ol type="a"> <li>PMT Trans-Gain 225V <li>PMT1-Range:500-550-Gain 550V</li> <li>PMT3-Range:750-800-Gain 600V</li></ol> <li>642 Laser-8.00%</li> <ol type="a"> <li>PMT Trans-Gain 300V <li>PMT1-Range:500-550-Gain 6000V</li> <li>PMT3-Range:750-800-Gain 550V</li></ol> </ol></ol> [[Category:PosSeq]] [[Category:20170706]]
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