Preparation of Competent XLI-Blue MRF' Cells

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Preparation of Competent XLI-Blue MRF' Cells

Preparation of Competent XLI-Blue MRF' Cells

Procedure:

1) Streak XLI-Blue MRF' cells from frozen stock onto SOB agar plate. Incubate ON at 37°C.

2) 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).

3) Dispense cells into (2) 50 ml prechilled conicals. Cool cultures for 10 min on ice, then spin for 10 min at 2,000 rpm (Beckman GS6R centrifuge; 4°C). Pour off supernatant, then leave inverted for 1 min to remove all supernatant.

4) Resuspend pellets in 20 ml/tube of ice-cold transformation buffer by gentle vortexing. Place on ice for 10 min. Spin for 10 min 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.

5) Add 140 l/tube of DMSO. Mix gently by swirling and store on ice for 15 min. Add an additional 140 l/tube of DMSO. Mix gently by swirling and place on ice. Quickly dispense 300 l aliquots into prechilled sterile 1.5 ml screw top tubes. Immediately snap freeze by immersing in liquid N2. Store in locator until needed. Before use, thaw one aliquot to check for transformation efficiency using pUC18 (see Transformation of Competent Cells).

Reagents:

1) SOB Medium Combine: Tryptone 16 gm (Difco #0123-05-3) ,Yeast Extract 4 gm (Difco #0127-05-3), NaCl 0.4 gm (Fisher #S671-500;) ,Make up to 800 ml in ddH2O. Swirl to dissolve, then autoclave. Before use add MgCl2 (10 ml/l of 1 M) and MgSO4 (20 ml/l of 1M; note: require 20 mM MgSO4 for this SOB).

2) SOB Plate Combine: Tryptone 16 gm (Difco #0123-05-3) ,Yeast Extract 4 gm (Difco #0127-05-3), NaCl 0.4 gm (Fisher #S671-500.) ,Bacto Agar 16 gm (Difco#0140-01;), Make up to 800 ml in ddH2O. Swirl to dissolve, then autoclave. Before use add MgCl2 (10 ml/l of 1 M) and MgSO4 (10 ml/l of 1M).

3) 1 M MgSO4 Combine: MgSO47H2O 24.7 gm (Sigma#M-2773) Make up to 100 ml in ddH2O. Swirl to dissolve, then autoclave.

4) 1 M MgCl2 Combine: MgCl26H2O 20.33 gm (USB#18641) Make up to 100 ml in ddH2O. Swirl to dissolve, then autoclave.

5) 1 M KAc (pH 7.5) Combine: KC2H3O2 3.93 gm Dissolve in 36 ml of ddH2O; adjust pH to 7.5 with several drops of 2 M acetic acid (2 M acetic acid is 0.6 ml of glacial acetic acid [17.4 M; Sigma#A-6283] made up to 5 ml in ddH2O). Bring to 40 ml with ddH2O. Dispense in 4 ml aliquots and store at -20°C.

6) Transformation Buffer Combine: 1 M KAc, pH 7.5 4 ml MnCl24H2O 3.56 gm (Sigma#M-3634) CaCl22H2O 0.59 gm (Sigma#C-3306) KCl 2.98 gm (Sigma#P-9541) Hex.cob. Cl 0.32 gm Hexaminecobalt chloride (Sig.#H-7891) Glycerol 40 ml (Sigma#G-5516) Dissolve in 320 ml of ddH2O; adjust pH to 6.4 with 0.1 N HCl. Make up to 400 ml with ddH2O, filter sterilize and dispense in 40 ml aliquots. Store at 4 °C.

Link: http://people.virginia.edu/~gwl6s/protocols/competentcells.html