Zippy Transformation of Z-competent Cells
JM109 = #T3005 from Zymo Research

1) Thaw the competent cells on ice for 5 minutes. Each tube contains 50 µL of cells. You can use these straight, aliquot them into 2 tubes of 25 µL each, or dilute up to 10 fold with Z-competent 2X Stock Competent Buffer solution (T3001-3-30). Diluting up to 10 fold will work for ligations as well as direct transformations of minipreps.

2) Very gently, aliquot cells into smaller volumes (we have used as low as 20 µL of cells with 5 µL of ligation) using pre-chilled microfuge tubes.

3) Add 1 - 5 µL of ligation mixture (can go as high as 10 µL for larger volumes of cells).

4) Incubate on ice for 5 minutes.

5) Add SOC media with no antibiotic to a final volume of 60 - 100 µL. Spread cells onto plates containing ampicillin*. You must let this sit for 30 minutes in the liquid at 37 C before plating if the antibiotic is not ampicillin.

You're done already!!

You can use colony PCR to screen your results before doign a miniprep.

* If you are using amplicillin, you can plate directly. But if you are using a different antibiotic, you need to let the cells produce proteins for 30 minutes so incubate them at 37 C to accelerate protein production. We have found for chloramphenicol, use 25 µg/mL for pSB1C8 and 10 µg.mL for pSB4C8.

© Copyright 2012 Department of Biology, Davidson College, Davidson, NC 28035
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