User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/05/22: Difference between revisions

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# 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.
# 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.
# 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 suspention on shaking platform or rotating mixer at a slow setting for 20 min at room temperature.
# 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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5/22/2012

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

  1. Warm selection agar plates at 37°C.
  2. Incubate BL-21(DE3) and NEB-10B competent cells on ice just until thawed. Use 30 μL per ligation.
  3. 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)
  4. 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.
  5. Pipette the total volume of cells + ligation onto the agar; spread using sterile glass beads.
  6. 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.
  1. For the recombinant plasmids BD-112 and BD113, compare the plates to estimate the ratio of “ligation” colonies to “negative control” colonies.
  2. 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.
  3. 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).
  4. 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).
  5. Grow the cultures overnight in a shaking 37°C incubator.

BugBuster Protein Extraction

  1. Harvest cells from liquid culture by centrifugation at 16000 ×g for 10 minutes.
  2. 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.
  3. 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.
  4. 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.
  5. Incubate the cell suspension on shaking platform or rotating mixer at a slow setting for 20 min at room temperature.
  6. Remove insoluble cell debris by centrifucation at 16000 ×g for 20 minutes ate 4°C.
  7. Transfer the supernatant to a fresh tube and keep at -20°C.
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