Daniel:Notebook/ComboLock/2016-3-11: Difference between revisions
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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. | 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> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
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===Run Parameters=== | |||
*Line 1: ~1 mL oil; 1.5 psi | *Line 1: ~1 mL oil; 1.5 psi | ||
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#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. | #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. | #We did appear to get droplets for a short time before the aqueous line drained and the stopper started blowing. | ||
==Discussion== | ==Discussion== |
Revision as of 15:46, 14 March 2016
Droplet/Polymerization Test
Today Andrew and I tried out a new setup for the microfludic device. Here is an image of the new setup:
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 (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)
Reagent | Stock Conc | Final Conc | Volume (uL) |
Ammonium Persulfate | 5% | 0.2% (0.1%) | 40 |
TEMED | 100% | 0.2% (0.1%) | 2 |
dH20 | 958 | ||
Total | 1000 |
Acrylamide/Bis (Line 3)
Reagent | Stock Conc | Final Conc | Volume (uL) |
Acrylamide | 40% | 18% (9%) | 450 |
Acrylamide/Bis | 40% (19:1) | 1.9%:0.1% (0.95%:0.05%) | 50 |
dH20 | 500 | ||
Total | 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 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
Reagent | Stock Concentration | Final Concentration | Amount added |
Acrylamide | 40% | 3% | 3 mL |
Acrylamide:Bis (19:1) | 40% (38:2) | 0.95:0.05 (%) | 1 mL |
VA-044 | ----- | 0.25% (m/v) | 0.1 g |
PBS | 10X | 1X | 4 mL |
PFA | 16% | 0% | 0 mL |
nfH2O | ----- | ----- | 32 mL |
Total | 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.