PstI restriction enzyme

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	After the chromosomal DNA and vector pUC119 (TaKaRa, Kyoto, Japan) were digested by restriction endonucleases PstI (TaKaRa) and EcoRI (TaKaRa), they were ligated with a DNA ligation kit, version 2 (TaKaRa).

Protocol tips
After the chromosomal DNA and vector pUC119 (TaKaRa, Kyoto, Japan) were digested by restriction endonucleases PstI (TaKaRa) and EcoRI (TaKaRa), they were ligated with a DNA ligation kit, version 2 (TaKaRa).

Publication protocol

The Otcr determinant was cloned from chromosomal DNA, because Vibrio sp. no. 6 seemed not to possess a plasmid (data not shown). However, it is possible that there exists a low-copy-number plasmid that could not be detected by agarose gel electrophoresis and/or a megaplasmid, which might be removed with the chromosome during extraction. The chromosomal DNA was extracted from the bacteria as follows. The bacterial pellet was suspended in extraction solution (0.15 M NaCl, 0.1 M EDTA, 0.5 mg of RNase A per ml, and 0.5% sodium dodecyl sulfate [SDS]) and incubated at 65°C for 5 min. This solution was mixed with phenol saturated with 10 mM Tris-HCl (pH 8.0) plus 1 mM EDTA (TE) and centrifuged (17,000 × g) for 3 min. The DNA in the supernatant was extracted two times with TE-saturated phenol-chloroform-iso-amyl alcohol (25:24:1 [vol/vol/vol]) and with chloroform. Precipitated DNA with ethanol was resuspended in 50 μl of TE and stored at −20°C. After the chromosomal DNA and vector pUC119 (TaKaRa, Kyoto, Japan) were digested by restriction endonucleases PstI (TaKaRa) and EcoRI (TaKaRa), they were ligated with a DNA ligation kit, version 2 (TaKaRa). Escherichia coli JM109 was transformed and spread on Luria-Bertani (LB) medium containing 50 μg of ampicillin per ml, 10 μg of OTC per ml, 40 μg of 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) per ml, and 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). After three passages of candidate colonies, we obtained a viable transformant. This transformant possessed a 919-bp inserted fragment, and the recombinant plasmid was designated pOV. Growth of transformants (pOV/JM109) with the cloned fragment was examined with or without MgCl2. The results are shown in Fig. ​Fig.1.1. pOV/JM109 grew in the LB broth with 25 μg of OTC per ml (Fig. ​(Fig.1A),1A), but not in the broth without MgCl2 (Fig. ​(Fig.1B1B). The same growth profiles were observed in the case of cultures containing 12.5 μg of tetracycline per ml (data not shown). This suggests that pOV/JM109 is also a tetracycline resistance determinant acting in the same manner as OTC and that the resistance ability of the transformed E. coli is dependent on MgCl2 as well as Vibrio sp. no. 6. Furthermore, we have retransformed pOV to different host E. coli DH5α cells. The transformants (pOV/DH5α) showed resistance in the presence of 10 mM MgCl2, the same as in JM109 (data not shown). On the other hand, the transformant pUC119/DH5α could not grow on the plate with OTC, regardless of the presence and absence of MgCl2. This suggests that Tet 34 can be expressed in both strains JM109 and DH5α

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