Transformation of Compenent E. coli[Yale University]

互联网2008-07-08

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Preparation of Frozen Competent E. coli

1. For TG1 or DH5a cells, start with a colony from a minimal plate to ensure the presence of the F ' episome. Grow in 5 mls LB o/n (see note on medium below). Inoculate a large culture (500 mls is good) 1/100 with the o/n culture and grow to OD₅₅₀=~.45 (takes 2-3 hrs).

1.5 An alternative growth procedure that yields cells 10-100 fold more efficient for transformation:

streak cells onto a Y plate, and grow o/n.

pick a single fresh colony and inoculate 5 ml Y medium, grow 2h at 37℃ (to OD₅₅₀=~0.3).

inoculate 100-500 mls prewarmed Y medium and grow for a further 2-3 hours, until OD₅₅₀=~0.45. See note on Y medium below.

2. Chill 5' on ice, spin 6k, 5', 4℃. All further steps performed in cold room with chilled solutions and pipettes. Once cells are treated with salts, they must be handled as gently as possible if high efficiency is to be obtained.

3. Resuspend in 2/5 volume TfbI. Leave on ice 5'. Spin 6k, 5', 4℃.

4. Resuspend in 1/25 original volume TfbII. Leave on ice 15'.

5. Make 200 µl aliquots and freeze in liquid nitrogen. Store at -80℃.

Transformation

6. Thaw cells at room temp (or by holding between fingers), and put on ice. Use 100 µl/transformation. Add DNA and incubate on ice 30'. It is best to add <100ng DNA in <40 µl. However, ligations performed in 20 µl low melt agarose can be transformed by melting at 70℃, adding 80 µl 0.1M Tris pH 7.5, chilling on ice, and adding this to 100 µl cells.

7. Heat shock at 42℃, 90", return to ice for 1'-2'.

8. Add 1 ml LB, incubate 1 hr at 37℃.

9. Plate on L plates containing 50-100µg/ml carbenicillin. (Note: I prefer carbenicillin to the more widely used ampicillin. Carb. is much more stable; plates and media containing carb. can be kept indefinitely, whereas ampicillin is slowly hydrolysized so that solutions and plates can only be used for a couple of weeks after they are made. On the other hand, carb. is 15X more expensive.)

10. Expected efficiency: 5x106 cfu/µg supercoiled DNA using growth method 1, 2x108 cfu/µg using growth method 1.5. Cells seem to maintain excellent efficiency for at least 2 years when stored at -80℃. Some people make fresh competent cells every time they do a transformation under the mistaken impression that frozen cells don't work well; this practice appears to me to be a foolish waste of time.

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Y medium: 5g/l yeast extract

20g/l tryptone

5g/l MgSO₄₂O

dH₂O to 1 liter

adjust to pH 7.6 w/ NaOH

14g/l agar for plates (or none for liquid)

autoclave

Note: At Yale this protocol hasn't been working lately for TG1, although DH5 has been fine. Georgia P. has solved the problem by adding 20 mM Tris pH8 to the Y medium. Michael Koelle has reproduced this success.

TfbI:

30mM potassium acetate 1.5g

100mM RbCl₂ 6.0g

10mM CaCl_{2 2}0 0.74g

50mM MnCl₂ * 4H₂0 4.95g

Add glycerol to 15% (v/v) 75ml

H₂O to 500ml

pH to 5.8 with ~10% acetic acid (careful, only takes a few drops)

Do not overshoot - it ruins the solution. You cannot adjust back with KOH.

Sterilize by filtration

Tfb II:

10mM MOPS 0.2g