Yu:e coli transform

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(Added heat-sock protocol)
Current revision (10:48, 8 June 2010) (view source)
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#Thaw DH5α cell stock (100μL/transformation) on ice for 30 min
#Thaw DH5α cell stock (100μL/transformation) on ice for 30 min
#Add 5-10 μL of ligation mix or ≤100 ng Plasmid to thawed cells, '''mix gently''', and incubate on ice for at least 30 min.
#Add 5-10 μL of ligation mix or ≤100 ng Plasmid to thawed cells, '''mix gently''', and incubate on ice for at least 30 min.
-
##Turn on 42°C heat block/water bath
+
#Remove the cells from the ice and immediately place them in the 42°C heat block for 90 sec.
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#Remove the cells from the ice and immediately place them in the 42°C heat blockk for 90 sec.
+
#Place the tubes back on ice for 5 min.
-
#Place the tubes back on ince for 5 min.
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#Add 0.5 mL of SOC medium to the cells and incubate at 37°C with shaking/rocking for 1 hr.
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#Add 0.5 mL of LB medium to the cells and incubate at 37°C with shaking/rocking for 1 hr.
+
#Plate the desired amount of cells on the appropriate selective plates
#Plate the desired amount of cells on the appropriate selective plates
##The amount of cells to be plated depends on the competency of the cells and the amount of plasmid used.  Plating too many cells may result in crowding no isolates, it is usually best to plate different amounts on different plates (i.e., 100 μL and 200 μL, or something similar)
##The amount of cells to be plated depends on the competency of the cells and the amount of plasmid used.  Plating too many cells may result in crowding no isolates, it is usually best to plate different amounts on different plates (i.e., 100 μL and 200 μL, or something similar)
#Incubate the plates at 37°C of ~14-16 hr.
#Incubate the plates at 37°C of ~14-16 hr.
#Pick single isolates for freezer stock and plasmid confirmation (PCR or R.E. digest)
#Pick single isolates for freezer stock and plasmid confirmation (PCR or R.E. digest)

Current revision

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E. coli transformation

Heat Shock transformation

This protocol is for "normal competent" DH5α, refer to manufacturer's insert for use of high-efficiency/sub-cloning efficiency cells
  1. Thaw DH5α cell stock (100μL/transformation) on ice for 30 min
  2. Add 5-10 μL of ligation mix or ≤100 ng Plasmid to thawed cells, mix gently, and incubate on ice for at least 30 min.
  3. Remove the cells from the ice and immediately place them in the 42°C heat block for 90 sec.
  4. Place the tubes back on ice for 5 min.
  5. Add 0.5 mL of SOC medium to the cells and incubate at 37°C with shaking/rocking for 1 hr.
  6. Plate the desired amount of cells on the appropriate selective plates
    1. The amount of cells to be plated depends on the competency of the cells and the amount of plasmid used. Plating too many cells may result in crowding no isolates, it is usually best to plate different amounts on different plates (i.e., 100 μL and 200 μL, or something similar)
  7. Incubate the plates at 37°C of ~14-16 hr.
  8. Pick single isolates for freezer stock and plasmid confirmation (PCR or R.E. digest)
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