Matt:LabNotes/Polyacrylamide Gel Protocol: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>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
 
Line 2: Line 2:
*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==


*Bind-Silane ([http://www.gelifesciences.com/webapp/wcs/stores/servlet/ProductDisplay?categoryId=11093&catalogId=10101&productId=22074&storeId=11787&langId=-1 GE Website])
*10% BSA ([http://www.sigmaaldrich.com/catalog/product/sigma/a1595?lang=en&region=US Sigma Alrich Website]
*10% BSA ([http://www.sigmaaldrich.com/catalog/product/sigma/a1595?lang=en&region=US Sigma Alrich Website]
*Teflon Coated Slides ([https://www.emsdiasum.com/microscopy/products/preparation/slides.aspx Electron Microscopy Sciences, Cat #63415-32])
*40% Acrylamide
*Acetic Acid
*40% Acrylamide/Bis (19:1)
*Slide Holders
*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 "ABD mix" in a 0.5 ml pcr tube:
#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              A-B-D mix (FILTERED)
##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
  • 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]

  1. Turn on UV lamp in AirClean hood for 15 minutes.
  2. Prepare fresh "AB mix" in a 0.5 ml pcr tube:
    1. 90 uL IEF 40% Acrylamide (made from powder)
    2. 10 uL Acrylamide / Bis (19:1; 38%:2%)
  3. 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.
  4. 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.
    1. 5 mg APS →100 uL dH20
  5. Prepare fresh 5% TEMED (in fume hood)
    1. 2 uL TEMED → 38 uL dH20.
  6. Put a few drops of 10% BSA into a 1.5 mL tube (BSA should be stored at 4'C)
  7. 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.
    1. 50 uL AB mix (FILTERED)
    2. 4 uL 10% BSA
    3. 138 uL dH20
    4. 4 uL 5% TEMED
    5. 4 uL 5% APS
  8. Add