Difference between revisions of "E coli Chemical Transformations"
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**5 μl ligated DNA; <u>or</u> | **5 μl ligated DNA; <u>or</u> | ||
**2-5 μl [[Gibson assembly| Gibson assembled]] plasmid; <u>or</u> | **2-5 μl [[Gibson assembly| Gibson assembled]] plasmid; <u>or</u> | ||
− | **2- | + | **2-10 μl [[ Golden Gate | Golden Gate assembled]] plasmid. |
*Flick and place back on ice for 30 min | *Flick and place back on ice for 30 min | ||
− | *Heatshock the cells for | + | *Heatshock the cells for 60sec at 42°C |
*Place back on ice for 2 min | *Place back on ice for 2 min | ||
*Add 250μl 37°C [[SOC]] or [[LB]] (without antibiotic) | *Add 250μl 37°C [[SOC]] or [[LB]] (without antibiotic) | ||
− | *Incubate these tubes with shaking at 37°C for 1 hour. | + | *Incubate these tubes with shaking at 37°C for 1 hour for ampicillin marker or 75-90min for kanamycin. |
*Preheat [[LB]] plates with appropriate antibiotic (50 μl [[Ampicillin]] 100mg/ml stock or 25 μl [[Kanamycin stock|Kanamycin]] 50mg/ml stock) to 37°C. | *Preheat [[LB]] plates with appropriate antibiotic (50 μl [[Ampicillin]] 100mg/ml stock or 25 μl [[Kanamycin stock|Kanamycin]] 50mg/ml stock) to 37°C. | ||
** add 40 μl [[X-Gal]] (freezer 7 top drawer) when blue white screening (e.g. using TOPO cloning) is required | ** add 40 μl [[X-Gal]] (freezer 7 top drawer) when blue white screening (e.g. using TOPO cloning) is required |
Latest revision as of 10:02, 11 September 2019
Transformation of competent E. coli cells
We use OneShot TOP10 Chemically competent E. coli cells (comparable to DH5α) or home-made competent cells that are to be found in the -80°C freezer (Parsch freezer).
When using commercial cells, for each transformation, we use 25μl. Each tube in the -80 can thus be used for 2 transformations.
Do transformation using the long version of the manufacturer’s protocol:
- Thaw the cells on ice (and split over 2 tubes to get 25μl cells, do not pipette more than once as cells are very sensitive).
- Add DNA to the cells:
- 5 μl PCR product of an sgRNA cloned linear plasmid; or
- 5 μl ligated DNA; or
- 2-5 μl Gibson assembled plasmid; or
- 2-10 μl Golden Gate assembled plasmid.
- Flick and place back on ice for 30 min
- Heatshock the cells for 60sec at 42°C
- Place back on ice for 2 min
- Add 250μl 37°C SOC or LB (without antibiotic)
- Incubate these tubes with shaking at 37°C for 1 hour for ampicillin marker or 75-90min for kanamycin.
- Preheat LB plates with appropriate antibiotic (50 μl Ampicillin 100mg/ml stock or 25 μl Kanamycin 50mg/ml stock) to 37°C.
- add 40 μl X-Gal (freezer 7 top drawer) when blue white screening (e.g. using TOPO cloning) is required
- Spread the 100 μl cells on the preheated LB+amp plates and incubate overnight at 37°C.
- the rest of the cells can be left in the fridge to plate out the next day if needed (e.g. when too many or too few colonies were obtained).
- After one day the plates should show colonies