5/22/2012
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
- 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.
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.
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