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Document: Competent cells 2 | Last modified: November 10, 2005
Making Competent Bacteria For Transformation By The Inoue Method
by Wen Dong, Nov 05

This protocol yield competent E.coli with a transfromation rate of 1-3 x 10e8 / ug plasmid DNA. Also, see this protocol and Measuring transformation efficiency.

Reagents

1. Inoue transformation buffer

Prepare 0.5 M PIPES (pH 6.7) by dissolving 15.1 g of PIPES in 80 ml of pure H2O (Milli-Q, or equivalent). Adjust the pH of the solution to 6.7 with 5 M KOH, and then add pure H2O to bring the final volume to 100 ml.

Sterilize the solution by filtration through a disposable prerinsed Nalgene filter (0.45-um pore size). Divide into aliquots and store frozen at -20 deg C.

Prepare Inoue transformation buffer by dissolving following in 800 ml of pure H
2O and then add 20 ml of 0.5 M PIPES (pH 6.7). Adjust the volume of the Inoue transformation buffer to 1 liter with pure H2O:

MnCl2.4H2O - 10.88 g - 55 mM final
CaCl2.2H2O - 2.2 g - 15 mM final
KCl - 18.65 g - 10 mM final

2. E. coli bacteria (DH5-alpha strain; you may use others)

Pick a single bacterial colony from agar plate (2-3 mm size; 16-20 h growth at 37 deg), and transfer to 25 ml LB broth or SOB broth in a 250 ml flask. Incubate at 37 deg for 6-8 h with vigorous shaking at 250-300 rpm.

3. DMSO

4. SOB medium

Dissolve 20 g tryptone, 5 g yeast extract and 0.5 g NaCl in 950 ml deionized water. Add 10 ml of 250 mM KCl. Adjust pH to 7.0 using 5 N NaOH (~0.2 ml) and add water to 1 liter. Sterilize by autoclaving for 20 min on a liquid cycle. Before using, add 5 ml sterile 2 M MgCl2.

5. SOC medium

Add 20 ml of sterile 1 M glucose to a liter of sterile SOB.

Method

1. Inocluate 8, 4 and 2 ml of the bacterial culture (above) respectively in three 1 litre flasks containing 250 ml SOB. Incubate at room temperature (18-22 deg C) overnight with moderate shaking.
2. Measure OD at 600 nm the next day. If, or whenever, one of the 3 cultures reaches OD of 0.55, transfer that flask to an ice-water bath and chill for 10 min.
3. Spin down cells - 2500 g x 10 min at 4 deg (3900 rpm in Sorvall GSA rotor).
4. Remove as much of the supernatant as possible by decanting and vacuum-aspirating liquid drops from the centrifuge bottle wall, etc.
5. Resuspend cells by swirling in 80 ml ice-cold Inoue buffer.
6. Spin down cells and remove supernatant as before.
7. Resuspend cells in 20 ml ice-cold Inoue buffer by swirling, and add 1.5 ml DMSO. Swirl and keep on ice for 10 min.
8. Aliquot (50 ul) into chilled sterile microfuge tubes on ice, cap the tubes tightly and snap freeze by immersing in liquid nitrogen.
9. Store at -70 to -80 deg C.

Transformation method

1. Thaw an aliquot by warming in hand and moving to ice just before the thaw.
2. transfer the cells to a chilled 17 x 100 mm polypropylene tube (such as Falcon 2059). (Glass tubes lower transformation efficiency.)
3. Add up to 25 ng DNA (upto 5% of volume of cells), and swirl to mix before storing on ice for 30 min.
4. Transfer to a 42 deg water bath and hold for 90 sec.
5. Move to ice to chill for 1-2 min.
6. Add 800 ul SOC medium, warm to 37 deg in a water bath, and then move to a shaking incubator (less than 225 rpm) at 37 deg for 45 min.
7. Plate on agar plates with selection agent (such as ampicillin, depending on the selection resistance marker in the plasmid DNA).
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