Quick-Competent Bacteria
This method is for rapidly preparing and transforming E. coli strains with supercoiled plasmid. The efficiency is not high, but this is very convenient for transforming rarely used strains or for sequential transformation of multiple plasmids into strains that are hard to co-transform. The procedure given below is for small scale preparation of competent cells, which is adequate for 10 transformations.
For cloning, use the Inoue method
Contents
Section titled “Contents”1. Procedure
Section titled “1. Procedure”- Grow bacteria from a fresh single colony in ~5 ml LB with any required antibiotics to an optical density of 0.3-0.4 at 600 nm.
- Transfer 1 ml cells to an eppendorf tube and incubate on ice for 10 min. At the same time, pipet 1 ml TSS solution into a separate tube and incubate on ice.
- Pellet cells at 1000 x g for 10 min at 4 deg.
- Gently resuspend the cells in 950 uL ice cold TSS solution.
- Add 50 uL DMSO, mix gently, and incubate on ice for 10 min.
- Proceed with standard heat shock transformation, using 100 ul of cells for each transformation.
2. Reagents
Section titled “2. Reagents”- TSS Solution: 10% (w:v) PEG8000, 50 mM MgCl2 in LB medium, adjusted to pH 6.5. Sterile filter and store at 4 deg.