User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/05/22: Difference between revisions
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# Re-suspend the cell pellet in room temperature BugBuster Reagent (EMD kit# 71370) by pipetting or gentle vortexing. Using 5mL of reagent per gram of wet cell paste. | # Re-suspend the cell pellet in room temperature BugBuster Reagent (EMD kit# 71370) by pipetting or gentle vortexing. Using 5mL of reagent per gram of wet cell paste. | ||
# To improve protein extraction efficiency add 40 μL of Lysonase (cat# 71230, an optimized ready to use mix of rLysozyme Solution and Benzonase Nuclease) per gram wet cell paste. Keep the Lysonase tube on ice during the process. | # To improve protein extraction efficiency add 40 μL of Lysonase (cat# 71230, an optimized ready to use mix of rLysozyme Solution and Benzonase Nuclease) per gram wet cell paste. Keep the Lysonase tube on ice during the process. | ||
# Incubate the cell | # Incubate the cell suspension on shaking platform or rotating mixer at a slow setting for 20 min at room temperature. | ||
#Remove insoluble cell debris by centrifucation at 16000 ×g for 20 minutes ate 4°C. | |||
#Transfer the supernatant to a fresh tube and keep at -20°C. |
Revision as of 17:18, 24 May 2012
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5/22/2012PcTF Extraction from E-coli Transform bacteria with the ligated plasmids 30 minutes
Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of vector self-ligation or selection failure. Grow liquid cultures
BugBuster Protein Extraction
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