EXPOSURE TO RECOMBINANT DNA
Always wear gloves and lab coat when handling recombinant DNA or E. coli.
Waste must autoclaved prior to disposal.
1. Thaw competent DH5a on ice. (Need 50 or 100ul per transformation). Use ÒhomemadeÓ bugs or Gibco #18265-017 (subcloning efficiency) bugs.
2. Aliquot 50 or 100ul into pre-chilled tubes.
3. Add 1/5th of the ligation mix or 20 ng. (Usually this volume is 4ul. Should not exceed 10% of the volume of the bugs).
Incubate 10-60 minutes on ice.
4. Heat shock at 42oC for 45 seconds, put back on ice immediately
5. Add 900ul LB. Incubate 60 minutes @ 37oC.
6. Spin bacteria 5 minutes at high speed. Remove all but 100ul supernatant.
7. Resuspend bacteria with a pipette and plate 90ul or 10ul on LB agar plates with antibiotic of choice.
8. Incubate ON at 37oC in bacterial incubator
9. Pick single colonies with a sterile toothpick (do not pick unusually large or small colonies). Add the toothpick to 3ml of LB + antibiotic in a 15ml round-bottom tube.
10. Incubate ON in shaker
11. This ON culture can be used for minipreps, frozen (aliquot .85ml of the culture into a freezer vial, add 150ul sterile glycerol, store at –70), or used to innoculate a 250ml culture for a large scale prep (use 250ul).