Coenen Lab Protocols last update: Aug 2001

Transformation of E. coli

Day 1:
Check to make sure that you have the following ready for tomorrow (numbers refer
to 4 transformations, multiply accordingly if you are doing more at once):

1.) 200 µl of competent E. coli cells, e.g. JM109 from Promega, stored at –70 OC
2.) 4 sterile Eppendorf tubes

3.) reserve water bath or heat block

4.) plasmid DNA samples

5.) ~ 5ml of SOC medium, stored at 4 OC

6.) 8 LB plates containing appropriate antibiotic

Day 2:
1.) Bring water bath to 42 OC, bring shaking incubator to 37 OC, get ice bucket.

2.) Chill sterile Eppendorf tubes on ice.

3.) Thaw frozen competent cells on ice for 5 min.
4.) Gently mix thawed cells and transfer 50 µl to each chilled tube.
5.) Add 1-50 ng of DNA to each tube, quickly flick tubes several times.
6.) Ice for 10 min.
7.) Heat-shock at exactly 42 OC for 45-50 seconds (no more!!). Do not shake!

8.) Immediately ice cells for 2 min.

9.) Add 450 µl cold SOC medium. Shake at 300 rpm, 37 OC for 60 min.

10.) Label two LB-Ab plats for each transformation. Plate 5 and 50 µl of each
transformation mix on LB-Ab plates by spreading with s. Keep remainder of
trafo mixes at 4 OC over night.
11.) Let plates dry in sterile hood for a bit before transferring them to 37 OC
incubator.
Day 3:
12.) Check plates after 12 – 14 hours.
13.) If there are no colonies on either plate, briefly centrifuge remainder of the
transfer mix, remove most of the supernatant, resuspend pellet in the
remainder and plate the entire mix on another LB-Ab plate.
14.) If there are colonies, do minipreps and check plasmid identity by restriction
analysis. Plates will keep at 4 O
for a couple of weeks.