238
ANNETTE DEICHMANN AND KLAUS DEICHMANN
ldentification
of the recombinant clones
12. Incubate on ice for 30 min.
13. Place the tube in a water bath at 42°C for exactly 2 min (heat shock).
14. Add 1 ml of LB medium and incubate at 37°C in a shaker or shaking
water bath for 1 hr.
15. Spread different amounts of this culture (e.g. 50, 100 and 200 ~1) over
the surface of LB agar plates containing the appropriate antibiotic ( depending on the plasmid) to obtain single colonies. Store the remainder
of the culture at 4 oc for further plating if necessary.
16. As a negative control, spread 100 ~1 of the untransformed competent
cells on a LB agar plate containing the appropriate antibiotic.
17. Place the plates inverted in an incubator at 37°C and let the cells grow
overnight.
18. Pick several colonies, using sterile toothpicks, to both an ampicillin and
a tetracycline plate. Use a grid so that you can identify the colonies belonging together.
19. Incubate the plates inverted overnight at 37°C in an incubator. Recombinant cloneswill grow only on one plate (ampicillin or tetracycline,
depending on the plasmid).
Subprotocol 2
Transformation of Bacteria with Recombinant Plasmid by
electroporation (Dower 1988)
The electroporation apparatus used for this experiment can be purchased
from different companies. W e describe here the use of the Gene Pulser system from Bio-Rad-Laboratories. The recombinant DNA tobe transformed
must be in a low ionic-strength buffer such as TE. The ligation mixture,
which can be used directly for transformation following the ca++ -precipitation method, is not suitable for electroporation. To circumvent this difficulty different methods such as dilution (Willson 1988), precipitation (Zabarovsky 1990), dialysis (Jacobs 1990) or heat inactivation (Ymer 1991) have
been described. Recently a new method which combines several of these
steps has been reported (Kobori 1993).
ANNETTE DEICHMANN AND KLAUS DEICHMANN
ldentification
of the recombinant clones
12. Incubate on ice for 30 min.
13. Place the tube in a water bath at 42°C for exactly 2 min (heat shock).
14. Add 1 ml of LB medium and incubate at 37°C in a shaker or shaking
water bath for 1 hr.
15. Spread different amounts of this culture (e.g. 50, 100 and 200 ~1) over
the surface of LB agar plates containing the appropriate antibiotic ( depending on the plasmid) to obtain single colonies. Store the remainder
of the culture at 4 oc for further plating if necessary.
16. As a negative control, spread 100 ~1 of the untransformed competent
cells on a LB agar plate containing the appropriate antibiotic.
17. Place the plates inverted in an incubator at 37°C and let the cells grow
overnight.
18. Pick several colonies, using sterile toothpicks, to both an ampicillin and
a tetracycline plate. Use a grid so that you can identify the colonies belonging together.
19. Incubate the plates inverted overnight at 37°C in an incubator. Recombinant cloneswill grow only on one plate (ampicillin or tetracycline,
depending on the plasmid).
Subprotocol 2
Transformation of Bacteria with Recombinant Plasmid by
electroporation (Dower 1988)
The electroporation apparatus used for this experiment can be purchased
from different companies. W e describe here the use of the Gene Pulser system from Bio-Rad-Laboratories. The recombinant DNA tobe transformed
must be in a low ionic-strength buffer such as TE. The ligation mixture,
which can be used directly for transformation following the ca++ -precipitation method, is not suitable for electroporation. To circumvent this difficulty different methods such as dilution (Willson 1988), precipitation (Zabarovsky 1990), dialysis (Jacobs 1990) or heat inactivation (Ymer 1991) have
been described. Recently a new method which combines several of these
steps has been reported (Kobori 1993).