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=Droplet/Polymerization Test= [[Daniel:Notebook/ComboLock|Back to Calendar]] Today Andrew and I tried out a new setup for the microfludic device. Here is an image of the new setup: [[Image:MFSetup_20160311.jpg|800px]] The setup allows us to use reagent reservoirs without much loss. This also keeps air bubbles from the lines, and helps with the problem of fouling that occurs when air gets into the device. The setup uses a syringe as the reservoir, and plugs the top with a rubber stopper. Air is introduced by a needle poked through the top. ==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. ==Protocol 2-VA-044== Next we tried to do the experiment with the VA-044 thermal initiator. Since we are unsure of polymerization temperature with VA-044 we did our best to keep the experiment on ice. As expected, the pressures were much higher, especially in the oil, since viscosity increases with decreasing temperature. We used the [[Daniel:Notebook/ComboLock/Polyacrylamide#VA-044|VA-044 recipe]] from CLARITY for this experiment, with water substituted for the PFA. This is because there is no biological tissue to fix with the PFA, although this may decrease the overall monomer concentration available, which may lead to weaker gels. ===Solutions=== {| 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="113" height="40" | Reagent | width="88" | Stock Concentration | width="82" | Final Concentration | width="87" | Amount added |- style="font-size:12pt" | height="15" valign="bottom" | Acrylamide | align="center" align="center" valign="bottom" | 40% | align="center" align="center" valign="bottom" | 3% | align="center" valign="bottom" | 3 mL |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Acrylamide:Bis (19:1) | align="center" valign="bottom" | 40% (38:2) | align="center" valign="bottom" | 0.95:0.05 (%) | align="center" valign="bottom" | 1 mL |- style="font-size:12pt" | height="15" valign="bottom" | VA-044 | align="center" valign="bottom" | ----- | align="center" valign="bottom" | 0.25% (m/v) | align="center" valign="bottom" | 0.1 g |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | PBS | align="center" valign="bottom" | 10X | align="center" valign="bottom" | 1X | align="center" valign="bottom" | 4 mL |- style="font-size:12pt" | height="15" valign="bottom" | PFA | align="center" align="center" valign="bottom" | 16% | align="center" align="center" valign="bottom" | '''0'''% | align="center" valign="bottom" | '''0''' mL |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | ----- | align="center" valign="bottom" | ----- | align="center" valign="bottom" | 32 mL |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" valign="bottom" | | align="center" valign="bottom" | |style="font-weight:bold" align="center" valign="bottom" | 40 mL |} ===Run Parameters=== *Line 1: ~1 mL oil; 1.5 psi *Line 2: ~2 mL aqueous mix; 9 psi ===Results=== #The run did not last long, and again the aqueous phase drained quickly compared with the oil. #The pressure in the oil line (9 psi) was so high that it kept popping the stopper off the reservoir syringe. We will probably have to tape down the stopper in future iterations. #We did appear to get droplets for a short time before the aqueous line drained and the stopper started blowing. ==Discussion== The principle thing I have decided from today is that we need to be more quantitative regarding droplet formation. There is no real check at this point for what droplets we are making: we do not evaluate droplet size in a quantitative manner nor do we test for whether or not gel formation truly occurred. Without these two things, we are relying on observational evidence to direct our actions. I will spend some more time evaluating how to better assess these conditions. Additionally, I made several recommendations in the results sections that will potentially mitigate some of the problems with these experiments.
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