User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/23: Difference between revisions
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== | ==SDS-PAGE== | ||
#Our protein solution was collected from the cold room and transferred into a 50mL falcon tube labeled Purified Hb pH7, 7/14/14 | |||
#We will run a gel with the solution and past solution to check purity | |||
#The gel will be set up in the following order | |||
##Myoglobin and BSA ladder | |||
##Initial protein solution | |||
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==Gel Electrophoresis== | |||
We will be using a Bio-Rad Mini Protean system with pre-cast Mini Protean TGX gels. The manual for this system can be found [http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1658100.pdf here] We will be running SDS-PAGE followed by gel development with Coomassie Blue staining. | |||
Below is a short description of how we will proceed. Please refer to the manual for more detailed instructions | |||
# Prepare the Gel and Assemble the Electrophoresis Cell | |||
## Remove comb and tape from the gels | |||
## Rinse the wells with running buffer | |||
## Load 18uL of ladder and sample in the wells | |||
# Perform electrophoresis | |||
## Run for 30 minutes at 200V (I need to make sure our power source can do this) | |||
# Develop/Stain your gel | |||
## Place gel in Fixative Solution for 30 minutes | |||
## Place gel in Stain Solution for 1 hour | |||
## Place gel in Destain Solution for 15 minutes | |||
### Repeat this step with fresh destain solution 1 more time | |||
==Stock Solutions== | |||
#Protein ladder | |||
-1mg BSA and 1mg Myoglobin in 1mL water | |||
-Solution was diluted to 1/10 to be used in gel | |||
#1X Running Buffer | |||
-14.4g Glycine, 1g SDS, 3g Tris in 100mL H2O | |||
-Solution was diluted to 1L with water | |||
#Fixative Solution | |||
-40 Methanol, 10% Acetic Acid, 50% Water | |||
#Stain Solution | |||
-90% water, 10$ Acetic Acid and .0025% Commassie Brilliant Blue | |||
#Destain Solution | |||
-90% Water and 10% Acetic Acid | |||
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SDS-PAGE
Gel ElectrophoresisWe will be using a Bio-Rad Mini Protean system with pre-cast Mini Protean TGX gels. The manual for this system can be found here We will be running SDS-PAGE followed by gel development with Coomassie Blue staining. Below is a short description of how we will proceed. Please refer to the manual for more detailed instructions
Stock Solutions
-1mg BSA and 1mg Myoglobin in 1mL water -Solution was diluted to 1/10 to be used in gel
-14.4g Glycine, 1g SDS, 3g Tris in 100mL H2O -Solution was diluted to 1L with water
-40 Methanol, 10% Acetic Acid, 50% Water
-90% water, 10$ Acetic Acid and .0025% Commassie Brilliant Blue
-90% Water and 10% Acetic Acid
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