User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/05/23: Difference between revisions
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#Prepare 500mL 1X NuPAGE SDS running buffer | #Prepare 500mL 1X NuPAGE SDS running buffer | ||
#*25mL 20X MES Running Buffer (for very small proteins e.g. 13kD) or 20X MOPS Running Buffer (for medium-sized proteins e.g. 50-60kD). | #*25mL 20X MES Running Buffer (for very small proteins e.g. 13kD) or 20X MOPS Running Buffer (for medium-sized proteins e.g. 50-60kD). | ||
#* | #*475mL deionized water | ||
#Mix well. | #Mix well. | ||
#Set aside 100mL buffer for use in Upper (Inner) buffer chamber). | #Set aside 100mL buffer for use in Upper (Inner) buffer chamber). | ||
#*Add | #*Add 500μL NuPAGE Antioxidant immediately before running the gel. | ||
#*Mix well. | #*Mix well. | ||
===Sample preparation=== | ===Sample preparation=== | ||
#Wells can accommodate 25μL loading volume. | #Wells can accommodate 25μL loading volume. | ||
#Let sample buffer warm to room temperature. | #Let sample buffer warm to room temperature. | ||
#Each sample should contain | #Each sample should contain | ||
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Note that prestained molecular weight marker doesn't need any preparation. | Note that prestained molecular weight marker doesn't need any preparation. | ||
After the heating process keep the samples on the bench to cool down to room temperature. | |||
===Running the gel=== | ===Running the gel=== |
Revision as of 17:53, 24 May 2012
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5/23/2012Materials
ProcedureRunning buffer preparationDo this step first.
Sample preparation
Set up the gel apparatus during the 10 mins sample heating step. Note that prestained molecular weight marker doesn't need any preparation. After the heating process keep the samples on the bench to cool down to room temperature. Running the gel
Staining the gel
You have three options for staining at this point.
See Knight:NuPAGE electrophoresis/Gel drying for instructions on how to dry and preserve the gel.
Marker Sizes
Notes
Safety
See here for detailed safety information. References |