INSTRUCTOR'S ADVANCE PREPARATION GUIDE a
b
c
d
e. Place the plates upside down inside the incubator overnight at 37°C or at room temperature for 2–3 days if an incubator is not available. Use for transformation within 24–36 hours as bacteria must be actively growing to achieve high transformation efficiency. Delaying more than 36 hours will compromise transformation. DO NOT REFRIGERATE BEFORE USE. f.
E. coli forms off-white colonies that are uniformly circular with smooth edges. Avoid using plates with contaminant colonies.
3. Prepare pGLO plasmid Using a new sterile pipet add 250 µl of transformation solution into the vial of lyophilized pGLO plasmid DNA. Note that the quantity of DNA is so small that the vial may appear empty. Invert the vial to mix plasmid solution well and ensure that the DNA is dissolved and is not sticking to the cap. If possible store the hydrated DNA in a refrigerator. (Transformation solution is being used here because it is a handy, sterile and nuclease-free solution. Sterile water would work just as well.)
250 µl
Transformation solution
Lyophilized pGLO plasmid DNA
Advance Preparation Step 3: Before Transformation Laboratory 1. Aliquot solutions For each student workstation, aliquot 1 ml of transformation solution (CaCl2) and 1 ml of LB nutrient broth into separate color-coded 2 ml microtubes provided in the kit. If the LB nutrient broth is aliquoted 1 day prior to the lab it should be refrigerated. Label the tubes. 2. Set up workstations for transformation laboratory See page 11 for materials to be supplied at each workstation.
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