Matt:LabNotes/Polyacrylamide Gel Protocol: Difference between revisions
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>Mzcai (Created page with "=Polyacrylamide Gel Protocol= *Based on Dan's protocol he learned from Dr. Zhang's Harvard protocol ==Purpose== *Make gel to cover brain tissue...") |
>Mzcai mNo edit summary |
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*Based on [[Daniel:Protocols/SlidePAGE|Dan's protocol he learned from Dr. Zhang's Harvard protocol]] | *Based on [[Daniel:Protocols/SlidePAGE|Dan's protocol he learned from Dr. Zhang's Harvard protocol]] | ||
==Purpose== | ==Purpose and General Procedure== | ||
*Make gel to cover brain tissue section for DARTFISH to prevent tissue degradation | *Make gel to cover and mechanically stabilize brain tissue section for DARTFISH to prevent tissue degradation | ||
* | *Instead of using dessicated bind-silane treated slides we have to use vectabond-treated cover glass in a tissue culture dish with a hydrated tissue section | ||
**Due to thickness of culture dish (~1mm) no way to use coverslip to prevent exposure to air while making <1mm thick gel | |||
***Can try using mineral oil to cover instead | |||
*Not using DATD (cross-linker) in Acrylamide/Bis mix | |||
==Materials== | ==Materials== | ||
*10% BSA ([http://www.sigmaaldrich.com/catalog/product/sigma/a1595?lang=en®ion=US Sigma Alrich Website] | *10% BSA ([http://www.sigmaaldrich.com/catalog/product/sigma/a1595?lang=en®ion=US Sigma Alrich Website] | ||
* | *40% Acrylamide | ||
* | *40% Acrylamide/Bis (19:1) | ||
* | *Syringe and 0.22 micron filter | ||
*Ammonium persulfate (APS) | |||
*TEMED | |||
*Dessicator | *Dessicator | ||
==Protocol== | ==Protocol== | ||
#Turn on UV lamp in AirClean hood for 15 minutes. | #Turn on UV lamp in AirClean hood for 15 minutes. | ||
#Prepare fresh " | #Prepare fresh "AB mix" in a 0.5 ml pcr tube: | ||
##90 uL IEF 40% Acrylamide (made from powder) | ##90 uL IEF 40% Acrylamide (made from powder) | ||
##10 uL Acrylamide / Bis (19:1; 38%:2%) | ##10 uL Acrylamide / Bis (19:1; 38%:2%) | ||
Line 28: | Line 31: | ||
#Put a few drops of 10% BSA into a 1.5 mL tube (BSA should be stored at 4'C) | #Put a few drops of 10% BSA into a 1.5 mL tube (BSA should be stored at 4'C) | ||
#Prepare the gel-casting mix (200 uL total volume). Do not add APS until immedietely prior to casting the gels. This recipe is for a 10% gel with sufficient mix for at least 8 (identical) slides. | #Prepare the gel-casting mix (200 uL total volume). Do not add APS until immedietely prior to casting the gels. This recipe is for a 10% gel with sufficient mix for at least 8 (identical) slides. | ||
##50 uL | ##50 uL AB mix (FILTERED) | ||
##4 uL 10% BSA | ##4 uL 10% BSA | ||
##138 uL dH20 | ##138 uL dH20 | ||
##4 uL 5% TEMED | ##4 uL 5% TEMED | ||
##4 uL 5% APS | ##4 uL 5% APS | ||
#Add |
Latest revision as of 20:43, 10 February 2016
Polyacrylamide Gel Protocol[edit]
Purpose and General Procedure[edit]
- Make gel to cover and mechanically stabilize brain tissue section for DARTFISH to prevent tissue degradation
- Instead of using dessicated bind-silane treated slides we have to use vectabond-treated cover glass in a tissue culture dish with a hydrated tissue section
- Due to thickness of culture dish (~1mm) no way to use coverslip to prevent exposure to air while making <1mm thick gel
- Can try using mineral oil to cover instead
- Due to thickness of culture dish (~1mm) no way to use coverslip to prevent exposure to air while making <1mm thick gel
- Not using DATD (cross-linker) in Acrylamide/Bis mix
Materials[edit]
- 10% BSA (Sigma Alrich Website
- 40% Acrylamide
- 40% Acrylamide/Bis (19:1)
- Syringe and 0.22 micron filter
- Ammonium persulfate (APS)
- TEMED
- Dessicator
Protocol[edit]
- Turn on UV lamp in AirClean hood for 15 minutes.
- Prepare fresh "AB mix" in a 0.5 ml pcr tube:
- 90 uL IEF 40% Acrylamide (made from powder)
- 10 uL Acrylamide / Bis (19:1; 38%:2%)
- Using a 3-cc syringe and a 0.22 micron filter, filter ~200 uL of the ABD mix into a 1.5 mL microcentrifuge tube.
- Prepare fresh 5% APS (ammonium persulfate). Notably, the APS bottle should be stored in some sort of room-temperature dessicator. We just store it inside a large plastic screw-top container.
- 5 mg APS →100 uL dH20
- Prepare fresh 5% TEMED (in fume hood)
- 2 uL TEMED → 38 uL dH20.
- Put a few drops of 10% BSA into a 1.5 mL tube (BSA should be stored at 4'C)
- Prepare the gel-casting mix (200 uL total volume). Do not add APS until immedietely prior to casting the gels. This recipe is for a 10% gel with sufficient mix for at least 8 (identical) slides.
- 50 uL AB mix (FILTERED)
- 4 uL 10% BSA
- 138 uL dH20
- 4 uL 5% TEMED
- 4 uL 5% APS
- Add