User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/05/22: Difference between revisions
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''' | '''PcTF Exctraction from ''E-coli''''' | ||
# | # '''Transform bacteria with the ligated plasmids''' ''30 minutes'' | ||
"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. | |||
#'''Grow liquid cultures''' | |||
##For the recombinant plasmids BD-112 and BD113, compare the plates to estimate the ratio of “ligation” colonies to “negative control” colonies. | |||
Note: 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. | |||
## If the ratio is 10:1 or greater, great job! Pick 2 colonies for separate liquid cultures. Grow for 5 - 6 hours.<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 2 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 for 7 hours in a shaking 37°C incubator. |
Revision as of 13:57, 24 May 2012
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5/22/2012PcTF Exctraction from E-coli
"Plasmids: BD112 and BD113, E-coli strains: BL-21(DE3) and NEB-10B"
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: 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.
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