Can carbenicillin be substituted for ampicillin when selecting for the pGEM® Vectors?
Yes, the antibiotic carbenicillin can be substituted for ampicillin in antibiotic selection plates when E. coli cells such as JM109 cells (Cat.# L2001) are transformed with or carry any of our pGEM® Vectors. The β-lactamase (bla) gene, which confers resistance to ampicillin, also confers resistance to carbenicillin, a semi-synthetic ampicillin analog. In some cases, carbenicillin may be preferred, as it tends to be more stable than ampicillin (i.e., less likely to break down in the presence of β-lactamases). and can reduce the growth of satellite colonies on plates during long-term incubations (1). Dissolve carbenicillin in a 50% ethanol solution and use at a final concentration of 100µg/ml in solid media.
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LabFact #32
Vary the incubation time and temperature for successful ligation. Blunt-end ligations benefit from lower temperatures (4–16°C) and longer incubation times (4 hours to overnight). Cohesive-end ligations can generally be performed at higher temperatures (15–20°C) with shorter incubation times (3–16 hours).
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