Preparation of Competent XLI-Blue MRF’ Cells¶

Contributed by Gordon W. Laurie, School of Medicine, University of Virginia, United States

Preparation of Competent XLI-Blue MRF’ Cells (ref. p. 1.76 of Maniatis)

Streak XLI-Blue MRF’ (#1059; in Box 1A of Locator) cells from frozen stock onto Super Optimal Broth (SOB) agar plate. Incubate overnight at 37°C.
Transfer 4-5 colonies (1-2 mm diameter) into 1 ml of SOB containing 20 mM MgSO4.
Disperse by vortexing at moderate speed, then dilute culture in 100 ml of SOB containing 20 mM MgSO4 in a 1 l flask (use flask reserved for preparation of competent cells).
Grow to OD600 of 0.45 -0.5 (equals approx. 108 cells/ml; check OD600 every 30-60 min).
Dispense cells into (2) 50 ml prechilled conicals. Cool cultures for 10 minutes on ice.
Spin for 10 minutes at 2,000 rpm (Beckman GS6R centrifuge; 4°C).
Pour off supernatant, then leave inverted for 1 minute to remove all supernatant.
Resuspend pellets in 20 ml/tube of ice-cold transformation buffer by gentle vortexing. Place on ice for 10 minutes.
Spin for 10 minutes at 2,000 rpm (Beckman GS6R centrifuge; 4°C).
Pour off buffer. Invert 1 min to remove all buffer.
Resuspend in 4 ml/tube of ice-cold transformation buffer by gentle vortexing.
Add 140 µl/tube of DMSO. Mix gently by swirling and store on ice for 15 minutes.
Add an additional 140 µl/tube of DMSO. Mix gently by swirling and place on ice.
Quickly dispense 300 µl aliquots into prechilled sterile 1.5 ml screw top tubes. Immediately snap freeze by immersing in liquid N2.
Store until needed. Before use, thaw one aliquot to check for transformation efficiency using pUC18.

References¶