PcTF Extraction 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.
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.