• Competent cells are fragile and should be kept on ice and handled very gently
• Go to –80C Freezer and in box 3 get out a (~25ul) frozen aliquot of competent cells
• Place on ice and wait until it thaws on ice-usually no more than 5 minutes
• Place a round bottom 2059 tube on ice, add all of thawed cells to bottom of tube
• Add 1ul BME (1:10 dilution of stock pure beta mercapto ethanol), gently stir
• Add 1-4ul plasmid DNA, gently stir but do not pipette up and down
• Leave on ice 5-30 minutes, occasionally gently swirl the tube itself
• Get a water bath set at 42C
• Place cells in water bath for 30 sec, no more or no less
• Immediately place cells on ice for 2 minutes

• Add 250ul SOC broth, then shake at 37C for 1 hour
• Pour onto LB agar plate with appropriate antibiotic, spread with sterile loop
• For BL-21, next day take a loop full of colonies and innoculate 1 liter 2XY T broth

Miniprep XL-1 blue

• Next day, use a sterilized loop to pick one colony to place in one tube of Lbroth with
• appropriate antibiotic added, repeat for total 4 colonies
• Place into 37C shaker overnight
• Follow Qiagen protocol for mini-prep the next morning