User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/05/22: Difference between revisions
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#'''Grow liquid cultures''' | #'''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. | |||
##For the recombinant plasmids BD-112 and BD113, compare the plates to estimate the ratio of “ligation” colonies to “negative control” colonies. | ##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). | |||
## If the ratio is 10:1 or greater, great job! Pick 2 colonies for separate liquid cultures | |||
## Label 15 ml sterile culture tube(s) appropriately. Fill each tube with | |||
## 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). | ## 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 | ## Grow the cultures overnight in a shaking 37°C incubator. |
Revision as of 14:03, 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.
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