Preparation of competent E.coli K12 Strains

Bacterial cells, when treated with calcium chloride, are able to take up phage l(Mandel and Higa, 1970) or plasmid DNA (Cohen, Chang and Hsu, 1973).

Many variations on the basic transformation technique have been developed to attempt to optimise the efficiency of transformation.

The method described here gives >108 colonies/µg DNA in a range of host strains.

Reagents:

Y Broth: 2% (w/v) Bacto Tryptone

0.5% (w/v) Bacto Yeast Extract

0.4% (w/v) MgSO4

10 mM KCl

Adjust pH to 7.6 with KOH

TfBI: 100 mM RbCl2

50 mM Mn Cl2

30 mM KOAc

10 mM CaC l2

15 % (v/v) Glycerol

Adjust pH to 5.8 with 0.2M HOAc (If too much acetic acid is added inadvertantly, throw the solution away — do not use!)

Sterile-filter and store in the fridge.

TfBII: 10 mM MOPS pH 7.0 (adjust with NaOH)

10 mM Rb Cl2

75 mM Ca Cl2

15% Glycerol

Sterile-filter and store in the fridge.

 

Procedure:

  1. To prepare competent cells

Work under sterile conditions.

  1. Pick a single colony into 5 ml Y broth. Grow at 37°C with shaking to OD550 0.3. This takes about 2-2.5 hrs.

  2. Innoculate 100 ml Y broth prewarmed to 37°C with this culture. To ensure good growth divide the starting culture onto 2x 50 ml cultures in 250 ml sterilins. Grow for 2-2.5 hrs with vigourous shaking (shaker speed 250 rpm) to OD550 0.48. Check OD after one hour and then every 20 min. Cells should double every 20-30 min.

  3. Chill cells on ice. Transfer the suspension to 2x 50 ml conicals and spin in a prechilled rotor at 2500 rpm for 5 min at 4°C to pellet the cells.

  4. If possible carry out the subsequent steps in a cold room. Take up the cells with brief vortexing in ice-cold TfBI. Use 30 ml TfBI for 100 ml culture, i.e. 15 ml per tube. Combine in one tube and chill on ice. The time for which the cells should be incubated in this solution depends upon the strain.

    5. DH5a: Leave on ice for 20-30 min.

    BL21: Leave on ice for 45 min.

  5. Spin down cells as in (3).

  6. Resuspend the cells gently with a 5 ml sterile pipette in 4 ml ice-cold TfBII per 100 ml starting culture. Dispense 0.2 ml aliquots into 1.5 ml labelled cryotubes (Strain, competent, Date) on ice or if possible on dry ice. Store at —70°C.

 

Calculation of the Transformation Efficiency

Check the transformation efficiency of the competent cells by transforming them with an uncut plasmid and calculating cfu/µg DNA. The plasmid of choice, if available, can be pUC18, which is a small plasmid confering Ampicillin resistance, provided with the commercial XL1blue cells from Stratagene at a concentartion of 0.1 ng/µl.

Transform 100 µl competent cells with 0.1ng of uncut plasmid DNA as usual (20-30 min on ice, 45 sec 42°C heatshock, 2 min on ice). Add 900 µl of LB to the transformation reaction (0.1 ngDNA /ml) and incubate as usual for 60 min at 37°C at 180 rpm.

From this volume, a 1:10 dilution (0.01ng DNA/ml) with LB is made and 100 µl plated on two plates (0.001ng DNA/100 µl). If 200 colonies are obtained (average of two plates), what is the transformation efficiency?

200 cfu = 2 x 105cfu/ng = 2 x 108cfu/µg DNA

0.001ng