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==Protocol 1-APS/TEMED== First we tested the drop seq device using APS/TEMED initiaors. We have only two regulators, so the aqueous lines received the same pressure and the oil line received a different pressure. We loaded ~1 mL of APS/TEMED in line 2, ~1 mL of Acylamide/Bis in line 3, and ~1 mL of oil in line 1 ([[Daniel:Notebook/ComboLock/Devices#Drop Seq Device|Design specs for Drop Seq Device]]). These are considerably higher volumes than we will probably use with samples (~10 uL), but it allows for greater experiment time for testing. Everything was kept at room temperature. ===Solutions=== '''APS/TEMED''' (Line 2) {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="116" height="41" | Reagent | width="90" | Stock Conc | width="137" | Final Conc | width="85" | Volume (uL) |- style="font-size:12pt" | height="15" valign="bottom" | Ammonium Persulfate | align="center" valign="bottom" | 5% | align="center" valign="bottom" | 0.2% (0.1%) | align="center" valign="bottom" | 40 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | TEMED | align="center" valign="bottom" | 100% | align="center" valign="bottom" | 0.2% (0.1%) | align="center" valign="bottom" | 2 |- style="font-size:12pt" valign="bottom" | height="15" | dH20 | align="center" | | align="center" | | align="center" | 958 |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | Total | align="center" | | align="center" | |style="font-weight:bold" align="center" | 1000 |} '''Acrylamide/Bis''' (Line 3) {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="116" height="49" | Reagent | width="90" | Stock Conc | width="137" | Final Conc | width="85" | Volume (uL) |- style="font-size:12pt" | height="15" valign="bottom" | Acrylamide | align="center" align="center" valign="bottom" | 40% | align="center" | 18% (9%) | align="center" valign="bottom" | 450 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Acrylamide/Bis | align="center" valign="bottom" | 40% (19:1) | align="center" | 1.9%:0.1% (0.95%:0.05%) | align="center" valign="bottom" | 50 |- style="font-size:12pt" valign="bottom" | height="15" | dH20 | align="center" | | align="center" | | align="center" | 500 |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | Total | align="center" | | align="center" | |style="font-weight:bold" align="center" | 1000 |} ===Run Parameters=== Pressures: *Aqueous: 2 psi *Oil: 3 psi ===Results=== #The aqueous line, specifically the AB aqueous line (3), drained quickly compared with the other two. This may indicate a clog in the other two lines. Since the APS/TEMED is a fast reaction that begins immediately upon mixing, we suspected the clog may have to do with polymerization occurring within the device. We decided to try another run, this time using the thermal initiator VA-044 in order to control polymerization more effectively. #It did appear that we were getting droplets prior to this event, however.
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