Advantagen

Simple and Easy Transformation protocol

1. Thaw the super competent cells on ice.
2. Transfer 1-5 µl ligation reaction mixture to the competent cells and mix gently.
3. Incubate the tube on ice for 20-30 minutes.
4. Heat-shock the cells in a 42 °C water bath for 45 seconds.
5. Incubate the tubes on ice for 1-2 minutes.
6. Add 1ml pre-warmed SOC medium.
7. Incubate at 37 °C for 30-60 minutes with or without shaking.
8. Plate ~ 0.1ml of the transformation mixture on LB agar plate with appropriate antibiotic and with IPTG and X-gal applied on the surface of the plate if colour screen is desired.

For electroporation:

1. Thaw the electro-competent cells on ice. Cool the cuvettes (0.1cm gap) on ice.
2. Prepare sample DNA from ligation reaction by one of following methods. A). Precipitate DNA with ethanol and resuspend DNA in 10 µl dd-H 2O to a concentration of 20-100ng/ ml. Add1 ml of DNA to the competent cells, mix gently. Transfer cells/DNA to the cold cuvette. B). Dilute the ligation reaction 10 times with dd-H 2O. Transfer 1-5 µl of diluted DNA to the cells. C). Transfer 1 microliterµl of ligation mixture directly to the competent cells.
3. Place the cuvette in the cuvette holder and electroporate (1250 v- 2000 v) the cells according to the manual of your instrument.
4. Follow step 6 as chemical transformation

Genotype: F-/endA1 recA1 hsdR17 (r_k^–, m_k⁺) glnV44 gyrA96 relA1 supE44 ø 80lacZdlacΔ(lacZ)M15

Storage : Advantagen's supper competent cells are stable at -80° C for at least two months.