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=Drop Seq Test (Started [[Daniel:Notebook/ComboLock/2016-3-15|03/15/2016]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] After [[Daniel:Notebook/ComboLock/2016-3-15#Results|yesterday's results]] it would appear that we may have polyacrylamide droplets. However, I think the best test is a chemical one, so we'll be following the Macosko protocol for droplet breakage. This protocol uses Perfluoro-1-octanol to break apart droplets. If the droplets did form PA beads, then the octanol shouldn't have an effect. If they didn't the droplets should disappear. ==Protocol== #Wash 3X with Mineral Oil (no Span) ##Remove oil phase with a pipette, and add in ~500 uL mineral oil ##Pipette the oil and beads gently to mix ##Repeat 1.1 and 1.2 #Wash 3X with 6X SSC ##Remove supernatant and add 500 uL 6X SSC ##Pipette gently to mix ##Centrifuge for 1 min at 1000xg ##Repeat steps 2.1-2.3 #Add 600 uL of 2% Perfluoro-1-Octanol in 6X SSC (200 uL 20X SSC, 12 uL Perfluoro-1-Octanol, 388 uL ddH2O) ##Shake vigorously by hand for 20 seconds (seriously) ##Centrifuge for 1 min at 1000xg ##Remove supernatant and add an additional 500 uL 6X SSC ##Repeat 3.2 and 3.3 3 times #Add 200 uL 6X SSC #Mix well to get beads into solution, then take 15 uL and deposit on to a slide #Seal slide with nail polish and wait >30 minutes to dry ===Results=== <gallery heights=400px widths=400px perrow=2 caption="Images from DropSeq/VA-044 Test"> File:20160317_DropSeqTest.lif_Field_1_ch00.jpg|Field 1 File:20160317_DropSeqTest.lif_Field_2_ch00.jpg|Field 2 File:20160317_DropSeqTest.lif_Field_3_ch00.jpg|Field 3 File:20160317_DropSeqTest.lif_Field_3;_zplane_2_ch00.jpg|Field 3; different z plane </gallery> ==Discussion== Looking at the gallery images, I believe the small dots are beads. During the protocol, I checked at each stage to make sure that I still had a gel phase. The best way to do this, or so I have found, is to invert the tube after centrifuging, and see if some of the material remains at the top. Once you dilute the beads in aqueous solution you entirely lose the gel loading dye, which we had been using to better visualize the droplets. The bubbles are likely either air bubbles from putting the coverslip on the slide or leftover oil bubbles that were not washed out.
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