3. Chloramphenicol stock solution: weigh 340 mg of chloramphenicol and dissolve in 10 mL ethanol. Store at À20
C. The
final concentration is 34 mg/mL chloramphenicol.
4. Rifampicin stock solution: weigh 0.5 g of rifampicin and dissolve in 10 mL of dimethyl sulfoxide. Store frozen aliquots at
À20
C. The final concentration is 50 mg/mL rifampicin.
5. Tetracycline stock solution: weigh 75 mg of tetracycline and
dissolve in 10 mL of ethanol. Store at À20
C. The final
tetracycline concentration is 7.5 mg/mL.
6. Kanamycin stock solution: weigh 0.5 g of kanamycin, dissolve
in 10 mL of water and sterilize by filtration. Store frozen
aliquots at À20
C. The final kanamycin concentration is
50 mg/mL.
Fig. 3 Schematic representation of an electrophoresis gel run to separate the
DNA fragments obtained in the NotI–PteI digestion of the pTV1-derivative and
pGTEVΔNIb, and the Eco81I–ApaI digestion of the pTV3-derivative and
pGTEVΔNIb. The pTV1 derivative and the NotI–PteI digestion, and the pTV3
derivative and the Eco81I–ApaI digestion are used to build recombinant
viruses that express the foreign proteins from the amino terminal end and
from the internal part of the viral polyprotein, respectively. Migration of the
schematic DNA fragments have been arbitrarily adjusted for a foreign cDNA of
1000 bp. DNA fragments marked with a red ellipse must be cut from the gel and
the DNA eluted for ligation
306
Jose ´ -Antonio Daro ` s
C. The
final concentration is 34 mg/mL chloramphenicol.
4. Rifampicin stock solution: weigh 0.5 g of rifampicin and dissolve in 10 mL of dimethyl sulfoxide. Store frozen aliquots at
À20
C. The final concentration is 50 mg/mL rifampicin.
5. Tetracycline stock solution: weigh 75 mg of tetracycline and
dissolve in 10 mL of ethanol. Store at À20
C. The final
tetracycline concentration is 7.5 mg/mL.
6. Kanamycin stock solution: weigh 0.5 g of kanamycin, dissolve
in 10 mL of water and sterilize by filtration. Store frozen
aliquots at À20
C. The final kanamycin concentration is
50 mg/mL.
Fig. 3 Schematic representation of an electrophoresis gel run to separate the
DNA fragments obtained in the NotI–PteI digestion of the pTV1-derivative and
pGTEVΔNIb, and the Eco81I–ApaI digestion of the pTV3-derivative and
pGTEVΔNIb. The pTV1 derivative and the NotI–PteI digestion, and the pTV3
derivative and the Eco81I–ApaI digestion are used to build recombinant
viruses that express the foreign proteins from the amino terminal end and
from the internal part of the viral polyprotein, respectively. Migration of the
schematic DNA fragments have been arbitrarily adjusted for a foreign cDNA of
1000 bp. DNA fragments marked with a red ellipse must be cut from the gel and
the DNA eluted for ligation
306
Jose ´ -Antonio Daro ` s