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=Droplet Test 2= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Background Findings== After [[Daniel:Notebook/ComboLock/2016-3-11|Friday]]'s experiment, I have done some thinking, and also read the Macosko paper searching specifically for how they reviewed their droplets. According two the paper, I have noticed a few interesting points: #They evaluated the droplets by observing aliquots under an optical microscope with brightfield illumination (10X, I believe) ##This appears to have been done with respect to bead occupancy of the droplets, it is probably favorable to add in molecules/markers we can evaluate #They used a high speed camera for videos; depending on how ease of integration, we may choose to do this with every experiment or as necessary for production. Still, I think a camera will be necessary before long #Phase contrast will be important, especially in these early stages. Andrew and I have already ordered several phase contrast reagents, but they are not here yet. We may consider using gel loading dye, SYBR, or Alexa fluorophores in the meantime. Hopefully, this may help the droplets show up under a light microscope. #The Macosko paper mentions using Perfluoro-1-octanol to break the droplets. This protocol should probably be followed in order to test whether or not the droplets formed are true polyacrylamide gels or still in solution ==Test 1== We found binder clips of an appropriate size to hold in the stoppers. This lets us use the higher pressures necessary for running the oil at low temperature (~10 psi). We also used a new setup, which is shown below. The new setup incorporates an ice bath for the reagents as well as the chip (on a block). It is then run across a hot plate at ~37-40C, across a light microscope for viewing. Finally, the solution is deposited in tubes waiting in the rock bath, also set at 37C. [[Image:MicrofluidicSetup_20160314.jpg|800px]] We tried the VA-044 again using the [[Daniel:Notebook/ComboLock/Polyacrylamide#VA-044 (Tissue)|standard recipe]]. ===Run Parameters=== *Line 1: Oil (on ice); 12 psi; ~2 mL *Line 2: Aqueous (CLARITY; on ice); 2 psi; ~1 mL ===Results=== We did get droplet formation, although the droplets were aqueous and could easily be popped using a pipette tip or needle. Therefore, we went back to the drawing board and realized that the acrylamide concentration we were using was too low. The CLARITY solution uses PFA to crosslink the proteins and nucleotides to the hydrogel matrix. This means that without any cellular material we should use extra acrylamide to create stiffer gels. ==Test 2 (10% Acrylamide)== This was cancelled and moved to [[Daniel:Notebook/ComboLock/2016-3-15|tomorrow]]. We did, however, make up the 10% acrylamide/VA-044 solution (below) {| 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="142" height="30" | Reagent | width="98" | Stock Concentration | width="99" | Final Concentration | width="139" | Amount added |- style="font-size:12pt" | height="15" valign="bottom" | Acrylamide | align="center" align="center" valign="bottom" | 40% | align="center" align="center" valign="bottom" | 9% | align="center" valign="bottom" | 9 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="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | ddH2O | align="center" valign="bottom" | ----- | align="center" valign="bottom" | ----- | align="center" valign="bottom" | 26 mL |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | |style="font-weight:bold" align="center" valign="bottom" | 40 mL |}
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