Induction of the CopyControl (Epicentre) Fosmid Clones to High Copy Number


Fosmid clones can be induced to high copy number for elevated yields of DNA for sequencing, fingerprinting or other applications. The induction process can be performed in any culture volume. In brief, a 1ml culture will provide a sufficient amount of DNA for most applications. The following protocol applies to the procedure using 1, 5, and 50ml of starting culture

Preparation of the culture:

  1. Each clone containing the fosmid of interest should be inoculated in 5ml of LB medium + 12.5 μg/ml chloramphenicol, in a 15ml tube.
  2. The cultures should be grown overnight at 37 C with shaking. The overnight culture will be used as inocula for the copy number amplification procedure.
  3. Using the following table, the appropriate volumes of LB + chloramphenicol, overnight expression culture and CopyControl induction solution (Epicentre) may be added to the desired volume of induction culture. Please ensure that the volume of tube or flask facilitates a good surface area for adequate aeration. For example, a 1.5 ml tube would be suitable for 1ml of induction culture.
  4.  

    Total volume of clone induction culture

    Volume of fresh LB + chloramphenicol (12.5 μ g/ml)

    Volume of overnight 5 ml culture

    Volume of 1000 × CopyControl Induction solution

    1 ml 800 μl 200 μl 1 μl
    5 μl 4.5 ml 500 μl 5 μl
    50 ml 45 ml 5 ml 500 μl

     

  5. Incubate the tubes at 37 C with vigorous shaking for 5 hours. To maximise the aeration of 1ml cultures, tape the 1.5 tubes horizontally to the shaking platform.
  6. The cells can be centrifuged and the DNA can be purified using standard laboratory methods.