Aim: To transform plasmid DNA to competent E. Coli cells
Material and Reagents
- LB medium
Ø 1% peptone
Ø 1% NaCl
Ø 0.5% Yeast Extract
- LB Agar plates with appropriate antibiotic.
- 1.5 mL microfuge tubes
- 42° C water bath
- Ice
- 37° C shaker
Procedure:
1.
Take out the competent cells from -800C
freezer.
2.
Thaw competent
cells (20–200µL per vial) on ice.
3.
Add max. 1 to 2µL
of a target plasmid (Commercial).
4.
Mix very gently.
5.
Incubate the tubes
on ice for 30 min
6.
Heat shocks the
cells for 90 sec at 42°C
7.
Place the tubes
immediately on ice for at least 2 min
8.
Add 1 mL of LB
medium to each tube.
9.
Incubate for 1
hour at 37°C and shake vigorously
10.
Spin down briefly
and remove most supernatants
11.
Resuspend cell
pellet with the rest LB medium in the tube by pipetting
12.
Plate out the
suspension on a LB agar plate containing the appropriate antibiotic.
13.
Incubate the
plates overnight at 37°C.
Theory video: https://youtu.be/7Ul9RVYG5CM
Practical video: https://youtu.be/1PqWnu2uBrY
Reference: https://www.addgene.org/protocols/bacterial-transformation/
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