15 Cloning Vectors
253
of TE buffer and stirr with a magnetic stirrer. Change the TE buffer 3
tim es.
26. Remave the DNA from the dialysis bag and determine the DNA concentration with the spectrophotometer at an OD260 (dilute an aliquot of
the DNA solution 1/20).
27. If you wish a higher DNA concentration, precipitate the DNA:
- In Eppendorf tubes mix the DN A solution with 2 1/2 volumes of 95o/o
ethanol and 1/10 volume of 3 M Na-acetate (pH 7.0).
- Incubate at -20°C for 1-2 hrs.
- Centrifuge at 4°C in the Eppendorf centrifuge for 10 min.
- Discard the supernatant carefully and wash the pellet with 70o/o ethanol (add 1 ml of 70o/o ethanol, vortex).
- Centrifuge at 4°C in the Eppendorf centrifuge for 5 min.
- Discard the supernatant and let the pellet dry in the vacuum evaporator.
- Dissalve the pellet in the desired volume of TE buffer.
References
Appleyard R K (1954) Segregation of new lysogenic types during growth of a doubly
lysogenic strain derived from Escherichia coli Kl2. Genetics 39:440-452
Bachmann B J (1972) Pedigrees of some mutant strains of Escherichia coli K12. Bacteriological Reviews 36:525-557
Bachmann B J, Low K B (1980) Linkage map of Escherichia coli K12, Edition 6. Microbiological Reviews 44:1-56
Birnboim H C (1983) A rapid alkaline extraction method for the isolation of plasmid
DNA. Methods Enzymol 100:243-255
Birnboim H C, Doly J ( 1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513-1523
Bolivar F, Rodriguez R L, Greene P J, Betlach M C, Heyneker H L, Boyer H W (1977)
Construction and characterization of new cloning vehicles. Il. A multipurpose cloning system. Gene 2:95-113
Boyer H W, Roulland-Dussoix D (1969) A complementation analysis of the restriction
and modification of DNA in Escherichia coli. J Mol Biol41:459-472
BRL-focus (1980) M13 Cloning/Didesoxy-sequencing manual
Collins J, Hohn B ( 1979) Cosmids: A type of plasmid gene-cloning vector that is packable
in vitro in bacteriophage Iambda heads. Proc Natl Acad Sei U.S.A. 75:4242-4246
Dower W J, Miller J F, Ragsdale C W (1988) High efficiency transformation of E.coli by
high voltage electroporation. Nucleic Acids Res 16:6127-6145
Frischauf A-M, Lehrach H, Poustka A, Murray N J (1983) Lambda replacement vectors
carrying polylinker sequences. J Mol Biol 170:827-842
253
of TE buffer and stirr with a magnetic stirrer. Change the TE buffer 3
tim es.
26. Remave the DNA from the dialysis bag and determine the DNA concentration with the spectrophotometer at an OD260 (dilute an aliquot of
the DNA solution 1/20).
27. If you wish a higher DNA concentration, precipitate the DNA:
- In Eppendorf tubes mix the DN A solution with 2 1/2 volumes of 95o/o
ethanol and 1/10 volume of 3 M Na-acetate (pH 7.0).
- Incubate at -20°C for 1-2 hrs.
- Centrifuge at 4°C in the Eppendorf centrifuge for 10 min.
- Discard the supernatant carefully and wash the pellet with 70o/o ethanol (add 1 ml of 70o/o ethanol, vortex).
- Centrifuge at 4°C in the Eppendorf centrifuge for 5 min.
- Discard the supernatant and let the pellet dry in the vacuum evaporator.
- Dissalve the pellet in the desired volume of TE buffer.
References
Appleyard R K (1954) Segregation of new lysogenic types during growth of a doubly
lysogenic strain derived from Escherichia coli Kl2. Genetics 39:440-452
Bachmann B J (1972) Pedigrees of some mutant strains of Escherichia coli K12. Bacteriological Reviews 36:525-557
Bachmann B J, Low K B (1980) Linkage map of Escherichia coli K12, Edition 6. Microbiological Reviews 44:1-56
Birnboim H C (1983) A rapid alkaline extraction method for the isolation of plasmid
DNA. Methods Enzymol 100:243-255
Birnboim H C, Doly J ( 1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513-1523
Bolivar F, Rodriguez R L, Greene P J, Betlach M C, Heyneker H L, Boyer H W (1977)
Construction and characterization of new cloning vehicles. Il. A multipurpose cloning system. Gene 2:95-113
Boyer H W, Roulland-Dussoix D (1969) A complementation analysis of the restriction
and modification of DNA in Escherichia coli. J Mol Biol41:459-472
BRL-focus (1980) M13 Cloning/Didesoxy-sequencing manual
Collins J, Hohn B ( 1979) Cosmids: A type of plasmid gene-cloning vector that is packable
in vitro in bacteriophage Iambda heads. Proc Natl Acad Sei U.S.A. 75:4242-4246
Dower W J, Miller J F, Ragsdale C W (1988) High efficiency transformation of E.coli by
high voltage electroporation. Nucleic Acids Res 16:6127-6145
Frischauf A-M, Lehrach H, Poustka A, Murray N J (1983) Lambda replacement vectors
carrying polylinker sequences. J Mol Biol 170:827-842