Induction of the CopyControl (Epicentre) C.
elegans 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:
- Each clone containing the fosmid of interest should be
inoculated in 5ml of LB medium + 12.5 g/ml chloramphenicol, in a
15ml tube.
- 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.
- 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.
|
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
|
- 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.
- The cells can be centrifuged and the DNA can be purified using
standard laboratory methods.