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Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. IV. The ligation of the bluescript vector with the inserts

100%

Borowicz B. P.

Journal of Plant Protection Research

|

1999

|

tom 39

|

nr 1

The technology used for the cloning of the open reading frame (ORF) I and II reflecting DNA fragments of the I-18 C gene in regard to the ligation reactions of the bluescript vector with the inserts, is presented. The main steps include: the calculation of the molar ratio of the bluescript plasmid to the ORF I and II reflecting DNA fragments as the inserts, the ligation reaction, the transformation of the E. coli cells of the XL- 1 strain with the ligation reactions products and growing of the transformed bacterial cells.

2

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. V. Identification of the bacterial colonies, containing the recombinant bluescript plasmids

100%

Borowicz B. P.

Journal of Plant Protection Research

|

1999

|

tom 39

|

nr 1

The technology applied for the cloning of the open reading frame (ORF) I and II reflecting DNA fragments in regard to the identification of the bacterial colonies, of E. coli XL-1 strain, containing the recombinant bluescript plasmids, is described. The particular steps include: the α-complementation test, growing of the preliminary selected bacterial colonies in culture, isolation of the plasmids from these colonies, the Nde I and Bam HI digestion of the plamids mini-preparations, analysis of the digestion products and the identification of the bacterial colonies, containing the bluescript plasmids with the cloned inserts by the agarose gel electrophoresis (2% gel, 1 x TBE buffer).

3

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. VII. The preparation of the inserts and the translational vector - pET-3a for ligation

100%

Borowicz B. P.

Journal of Plant Protection Research

|

1999

|

tom 39

|

nr 1

The technology applied for the cloning of the open reading frame (ORF) I and II reflecting DNA fragments in regard to the preparation of the inserts and the translational vector pET-3a for ligation, is described. The main steps of the preparation of the inserts include: digestion of the isolated recombinant bluescript plasmids, carrying the inserts, with the Nde I and Bam HI, purification and isolation of the inserts after the digestion, by the preparative agarose gel electrophoresis (2% gel, 1 x TAE buffer) and then by the centrifugation through the filtration device of the isolated DNA fragment from the gel and also estimation of the inserts concentration by the agarose gel electrophoresis (2% gel, 1 x TBE buffer). The main steps of the preparation of the pET-3a vector included: transformation of the competent E. coli cells of the XL-1 strain with the pET-3a, growing of the transformed bacteria on agar plates (with ampicillin) and then growing of the isolated transformed bacterial colonies in culture to finally isolate the plasmid, digestion of the plasmid with the Nde I and Bam HI, purification of the isolatcd plasmid, after the digestion, by the preparative agarose gel electrophoresis (0.8% gel, 1 x TAE buffer), centrifugation through the filtration device of the isolated DNA fragment from the gel, phenol extraction of the isolated plasmid, estimation of the concentration of the plasmid by the agarose gel electrophoresis (0.8% gel, 1 x TBE buffer).

4

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. VI. The DNA sequencing of the cloned inserts

100%

Borowicz B. P.

Journal of Plant Protection Research

|

1999

|

tom 39

|

nr 1

The technology used for the cloning of the open reading frame (ORF) I and II reflecting DNA fragments in regard to the DNA sequencing of the cloned inserts, is presented. The object of the DNA sequencing of the cloned inserts, was to finally confirm and prove the sequence of the inserts as the sequence of the ORF I and II reflecting DNA fragments of the I-18 C gene of Chironomus tentans.

5

Cloning of the fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. IX. The effects of the cloning experiments

100%

Borowicz B. P.

Journal of Plant Protection Research

|

1999

|

tom 39

|

nr 1

The goal of these experiments was to clone the open reading frame (ORF) I and II reflecting DNA fragments of the I-18 C gene of Chironomus tentans into the pET-3a vector to translate them in the T7 RNA polymerase / promoter system in BL21(DE3) cells of E. coli. This goal has been achieved and the results of the cloning experiments are presented and discussed.

6

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. VIII. The ligations of the inserts with the pET-3a vector and the identification of the bacterial colonies, containing the recombinant plasmids

80%

Borowicz B. P.

Journal of Plant Protection Research

|

1999

|

tom 39

|

nr 1

The technology used for the cloning of the open reading frame (ORF) I and II reflecting DNA fragments in regard to the ligations of the inserts with the pET-3a vector and the identification of the bacterial colonies, containing the recombinant plasmids, is presented. The main steps include: calculation of the molar ratios of the vector to insert DNA, the ligation reactions, transformation of the competent E. coli cells of the XL-1 strain, isolation of the plasmids, digestion of the plasmid preparations with the Nde I and Bam HI to indicate the presence of the inserts cloned into the plasmids, using the agarose gel electrophoresis (2% gel, 1 x TBE buffer) of the plasmids samples, with the released inserts, after the digestion, transformation of the competent BL21 (DE3) cells of E. coli with the isolated recombinant plasmids, identification of the bacterial cells, carrying the pET-3a plasmids with the cloned inserts and the storage of these cells.

Ograniczanie wyników

6 Journal of Plant Protection Research

6 Borowicz B. P.

6 1999

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. IV. The ligation of the bluescript vector with the inserts

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. V. Identification of the bacterial colonies, containing the recombinant bluescript plasmids

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. VII. The preparation of the inserts and the translational vector - pET-3a for ligation

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. VI. The DNA sequencing of the cloned inserts

Cloning of the fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. IX. The effects of the cloning experiments

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. VIII. The ligations of the inserts with the pET-3a vector and the identification of the bacterial colonies, containing the recombinant plasmids