Daniel:Notebook/ComboLock/Polyacrylamide: Difference between revisions

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>Djacobse
(Created page with "=Polyacrylamide Gel Recipes for Combo Lock= Back to Notebook PA gel creation recipes. ==APS and TEMED== Based on the [[http://arep.med.harvar...")
 
>Djacobse
 
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Based on the [[http://arep.med.harvard.edu/polony/polony_protocols/bind_silane.htm protocol] from George Church's lab for PA Gel creation on glass slides.
Based on the [[http://arep.med.harvard.edu/polony/polony_protocols/bind_silane.htm 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.
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.


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==VA-044==
'''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.
 
{| 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" | &nbsp;
| align="center" | &nbsp;
| align="center" | 958
 
|- style="background-color:#D9D9D9;font-size:12pt"  valign="bottom"
|style="font-weight:bold" height="15" | Total
| align="center" | &nbsp;
| align="center" | &nbsp;
|style="font-weight:bold" align="center" | 1000
 
|}
 
==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 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>
|- 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" | 4%
| align="center" valign="bottom" | 10 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" | 22 mL
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Total
| align="center"  valign="bottom" | &nbsp;
| align="center"  valign="bottom" | &nbsp;
|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" | &nbsp;
| align="center" align="center" valign="bottom" | &nbsp;
|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]

Back to Notebook

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