Preparation of competent cells.

Monday, June 07, 2004

Description
This rubidium chloride protocol gives better transformation efficiencies than calcium chloride procedures for most strains.

Procedure
1) Innoculate a single colony from an LB plate in 2.5 ml of LB medium. Incubate overnight at 37 degrees with shaking (225 rpm).

2) Innoculate 250 ml of LB with 2.5 ml (1:100) of the overnight culture. Add 5 ml of 1M MgSO4 (20 mM final conc.). Grow cells in 1L flask until A600 is 0.4 to 0.6 (approx. 5-6 hours).

3) Pellet cells, centrifuge at 4,500 g for 5 mins at 4 degrees.

4) Gently resuspend cells in 0.4 volumes (of original culture vol) of ice cold TFB1. For a 250 ml sub culture, use 100 ml of TFB1.

For remaining steps keep cells on ice and chill all equipment.

5) Incubate resuspende cells on ice for 5 mins at 4 degrees.

6) Pellet cells by centrifugation, 4,500 g for 5 mins at 4 degrees.

7) Gently resuspend cells in 1/25 volume of ice cold TFB2. For 250 ml culture use 10 ml.

8) Incubate cells on ice for 15-60 mins and then aliquote 200 ul/tube for storage at -70 degrees. Quick freeze tubes in a dry ice/isopropanol bath. Most cells prepared this way are stable for at least three months.

Recipes
TFB1: 30mM potasium acetate, 10mM CaCl2, 50 mm MnCl2, 100Mm RbCLl 15 % Glycerol. Adjust pH to 5.8 with 1 M acetic acid, filter sterilize (0.2 Um) and store at room temp.

TFB2: 10mM MOPS or DIPES(pH 6.5), 75 mM CaCl2, 10mM RbCl, 15 % glycerol. Adjust pH to 6.5 with 1M KOH. Filter sterilize (0.2 uM) and store at room temp.

Supplies

Tips

(责任编辑：泉水)

搜索

热门标签:

Preparation of competent cells.