Daniel:Notebook/ComboLock/Polyacrylamide: Difference between revisions
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==VA-044== | ==VA-044 (Tissue)== | ||
VA-044 is a thermal initiator for creating free radicals that is principally used in the [http://www.nature.com/nature/journal/v497/n7449/full/nature12107.html | VA-044 is a thermal initiator for creating free radicals that is principally used in the [http://www.nature.com/nature/journal/v497/n7449/full/nature12107.html CLARITY protocol]. It is recommended to keep the mixed solution at 4C until activation. Activation temp is approximately 37C. I can actually find little information on VA-044 at the moment, quite a bit of my knowledge on it is empirical. It comes in a powder. | ||
{| 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> | ||
|- style="background-color:# | |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | ||
| width="113" height="40" | Reagent | | width="113" height="40" | Reagent | ||
| width="88" | Stock Concentration | | width="88" | Stock Concentration | ||
Line 136: | Line 136: | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | | ||
|style="font-weight:bold" align="center" valign="bottom" | 40 mL | |||
|} | |||
==VA-044 (No-Tissue)== | |||
The VA-044 solution made for no cells (empty droplets) incorporates 10% acrylamide instead of 4%. This higher concentration is to make up for the lack of nucleotides and protein which are used in the crosslinking process via formaldehyde. The percentage is based on the APS/TEMED receipe above. | |||
{| 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 | |style="font-weight:bold" align="center" valign="bottom" | 40 mL | ||
|} | |} |
Latest revision as of 23:27, 14 March 2016
Polyacrylamide Gel Recipes for Combo Lock[edit]
PA gel creation recipes.
APS and TEMED[edit]
Based on the [protocol from George Church's lab for PA Gel creation on glass slides.
This recipe is done on the Drop Seq device and involves using APS and TEMED, which is a fast reaction done at room temperature. As such, you make two different solutions, one with the acrylamide and bis monomers, the other with APS and TEMED. These are combined at the first cross-junction in the drop seq device.
Acrylamide/Bis Solution
This solution should also include sample, when using a sample. Fill the rest (after acylamide, bis, and sample) with water.
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 |
APS/TEMED Solution
Mixed at the first cross junction in the Drop Seq device (must be added before oil/droplet formation). APS/TEMED is the initializer/accelerator, and therefore must be combined as late as possible before droplet formation to prevent clogging.
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 |
VA-044 (Tissue)[edit]
VA-044 is a thermal initiator for creating free radicals that is principally used in the CLARITY protocol. It is recommended to keep the mixed solution at 4C until activation. Activation temp is approximately 37C. I can actually find little information on VA-044 at the moment, quite a bit of my knowledge on it is empirical. It comes in a powder.
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% | 4% | 10 mL |
nfH2O | ----- | ----- | 22 mL |
Total | 40 mL |
VA-044 (No-Tissue)[edit]
The VA-044 solution made for no cells (empty droplets) incorporates 10% acrylamide instead of 4%. This higher concentration is to make up for the lack of nucleotides and protein which are used in the crosslinking process via formaldehyde. The percentage is based on the APS/TEMED receipe above.
Reagent | Stock Concentration | Final Concentration | Amount added |
Acrylamide | 40% | 9% | 9 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 |
ddH2O | ----- | ----- | 26 mL |
Total | 40 mL |