User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/05/22: Difference between revisions
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'''PcTF | '''PcTF Extraction from ''E-coli''''' | ||
'''Transform bacteria with the ligated plasmids''' ''30 minutes'' | |||
<br>''Plasmids: BD112 and BD113, ''E-coli'' strains: BL-21(DE3) and NEB-10B'' | |||
# Warm selection agar plates at 37°C. | |||
# Incubate BL-21(DE3) and NEB-10B competent cells on ice just until thawed. Use 30 μL per ligation. | |||
# Add 30 μL thawed cells to the ligation reaction. Immediately place on ice and incubate for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp resistance) | |||
# Label the pre-warmed plates with the antibiotic name, strain name, ligation (e.g., "BB part A insert + BB part B vector"), your initials, and the date. | |||
# Pipette the total volume of cells + ligation onto the agar; spread using sterile glass beads. | |||
# Incubate overnight at 37°C to get colonies | |||
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. | 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''' | |||
*More than 100 ligation colonies on BD-112 and many colonies on BD-113 plate and no negative control colony were observed. The NEB-10B colonies are very tiny compare to BL-21 colones. | *More than 100 ligation colonies on BD-112 and many colonies on BD-113 plate and no negative control colony were observed. The NEB-10B colonies are very tiny compare to BL-21 colones. | ||
#For the recombinant plasmids BD-112 and BD113, compare the plates to estimate the ratio of “ligation” colonies to “negative control” colonies. | |||
# If the ratio is 10:1 or greater, great job! Pick 2 colonies (named as A and B) for separate liquid cultures.<br>''If the ratio is less than 10:1, pick more colonies or trouble shoot and repeat the ligation & transformation.'' | |||
# Label 15 ml sterile culture tube(s) appropriately. Fill each tube with 10 ml of LB growth medium + appropriate antibiotic (e.g., 100 μg/ml ampicillin). | |||
# Using a sterile pipette tip, touch the bacterial streak (or pick up a single colony) and put the tip into the LB medium (bacterial end down). | |||
# Grow the cultures overnight in a shaking 37°C incubator. | |||
'''BugBuster Protein Extraction''' | |||
#Harvest cells from liquid culture by centrifugation at 16000 ×g for 10 minutes. | |||
# Weigh the cell pellet. Weigh the same size empty well, zero the balance, remove the empty well and then weigh the well with cell pellet. | |||
# 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. | |||
# 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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