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

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=Date=
=5/22/2012=


'''List title'''
'''PcTF Exctraction from ''E-coli'''''
# List items
# '''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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5/22/2012

PcTF Exctraction from E-coli

  1. 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.

  1. Grow liquid cultures
    1. 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.

    1. If the ratio is 10:1 or greater, great job! Pick 2 colonies for separate liquid cultures. Grow for 5 - 6 hours.
      If the ratio is less than 10:1, pick more colonies or trouble shoot and repeat the ligation & transformation.
    2. 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).
    3. 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).
    4. Grow the cultures for 7 hours in a shaking 37°C incubator.
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