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

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'''PcTF Exctraction from ''E-coli'''''
'''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"
'''Transform bacteria with the ligated plasmids''' ''30 minutes''
## Warm selection agar plates at 37°C.
<br>''Plasmids: BD112 and BD113, ''E-coli'' strains: BL-21(DE3) and NEB-10B''
## Incubate BL-21(DE3) and NEB-10B competent cells on ice just until thawed. Use 30 μL per ligation.
# Warm selection agar plates at 37°C.
## 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)
# Incubate BL-21(DE3) and NEB-10B competent cells on ice just until thawed. Use 30 μL per ligation.
## 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.
# 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)
## Pipette the total volume of cells + ligation onto the agar; spread using sterile glass beads.
# 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.
## Incubate overnight at 37°C to get colonies
# 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.
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'''
'''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.  
*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.''
# 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).
# 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).
# 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.
# Grow the cultures overnight in a shaking 37°C incubator.

Revision as of 14:08, 24 May 2012

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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

  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.
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