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

• Autorzy: Borowicz B P
• Języki publikacji: EN

Abstrakty

EN

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.

PL

Przedstawiono technologię użytą do klonowania fragmentów DNA odzwierciedlających otwartą ramę odczytu I i II genu I-18 C Chironomus tentans w zakresie ligacji wstawek z wektorem pET-3a oraz identyfikacji kolonii bakteryjnych, zawierających rekombinacyjne plazmidy. Główne etapy obejmują: obliczenie stosunków molarnych wektora do DNA wstawki, reakcje ligacji, transformację komórek kompetentnych E. coli szczepu XL-1, izolację plazmidów, trawienie preparatów plazmidów Nde I i Bam Hl w celu wskazania obecności wklonowanych do plazmidów wstawek, uwolnionych z tych plazmidów poprzez trawienie wymienionymi enzymami restrykcyjnymi. Analizę produktów trawienia poszczególnych próbek plazmidów wykonano przy użyciu elektroforezy w żelu agarozowym (2% żel, 1 x stężony bufor TBE). Następne etapy to transformacja komórek kompetentnych E. coli BL21(DE3) preparatami wyizolowanych zrekombinowanych plazmidów pET-3a i identyfikacja komórek bakteryjnych, zawierających plazmidy pET-3a z wklonowanymi wstawkami oraz przechowywanie tych komórek.

Słowa kluczowe

EN

pET-3a vector; promoter system; cloning; I-18 C gene; T7 RNA polymerase; Chironomus tentans; polymerase chain reaction; expression; DNA fragment; recombinant plasmid; identification

• Wydawca: -
• Czasopismo: Journal of Plant Protection Research
• Rocznik: 1999
• Tom: 39
• Numer: 1
• Opis fizyczny: p.74-79

Twórcy

autor

Borowicz B P

• Institute of Plant Protection, Miczurina 20, 60-318 Poznan, Poland

Bibliografia

• 1. Borowicz B.P. 1995. Isolation of the DNA fragment reflecting the Open Reading Frame I of the I-18 C gene of Chironomus tentans by the Polymerase Chain Reaction. I. The technology for the preparation of the samples (in English). Prace Nauk. Inst. Ochr. Roślin 36 (1/2): 69-76.
• 2. Borowicz B.P. 1995. Isolation of the DNA fragment reflecting the Open Reading Frame I of the I-18 C gene of Chironomus tentans by the Polymerase Chain Reaction. II. The technology required to obtain the DNA fragment (in English). Prace Nauk. Inst. Ochr. Roślin 36 (1/2): 77-82.
• 3. Borowicz B.P. 1995. Isolation of the DNA fragment reflecting the Open Reading Frame I of the I-18 C gene of Chironomus tentans by the Polymerase Chain Reaction. III. The effect of the applied technologies (in English). Prace Nauk. Inst. Ochr. Roślin 36 (1/2): 83-93.
• 4. Borowicz B.P. 1996. Isolation of the DNA fragment reflecting the Open Reading Frame II of the I-18 C gene of Chironomus tentans by the Polymerase Chain Reaction. I. The technology for the preparation of the primers. J. Plant Protection Res., 37 (1/2): 138-144.
• 5. Borowicz B.P. 1996. Isolation of the DNA fragment reflecting the Open Reading Frame II of the I-18 C gene of Chironomus tentans by the Polymerase Chain Reaction. II. The technology for the preparation of the template. J. Plant Protection Res., 37 (1/2): 145-148.
• 6. Borowicz B.P. 1996. Isolation of the DNA fragment reflecting the Open Reading Frame II of the I-18 C gene of Chironomus tentans by the Polymerase Chain Reaction. III. The DNA amplification technology. J. Plant Protection Res., 37 (1/2): 149-155.
• 7. Borowicz B.P. 1996. Isolation of the DNA fragment reflecting the Open Reading Frame II of the I-18 C gene of Chironomus tentans by the Polymerase Chain Reaction. IV. The product of the applied technologies. J. Plant Protection Res., 37 (1/2): 156-164.
• 8. Borowicz B.P. 1996. Isolation of the DNA fragment reflecting the Open Reading Frame II of the I-18 C gene of Chironomus tentans by the Polymerase Chain Reaction. V. Different mechanisms of regulation regarding the Open Reading Frames. J. Plant Protection Res., 37 (1/2): 165-171.
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