26 pET-BccI

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	One Shot BL21(DE3) chemically competent E. coli bacteria (Invitrogen) were transformed, according to the manufacturer’s protocol, with 10 ng of each of the 26 pET-BccI plasmids carrying the BRP gene, 25 pET-BccI plasmids with the CAT gene and 22 pET-BccI plasmids carrying the CSFVRP gene. Two colonies from each transformation reaction were used to inoculate equal numbers of 3 ml LB/kanamycin pre-cultures, which were incubated overnight at 37°C and at 250 rpm. The following day, 15 ml cultures (LB/kanamycin) were inoculated with the appropriate volume from their corresponding pre-cultures in order to obtain an optical density (OD)600 value 0.1. The main cultures were incubated up to OD600 = 0.6 and then IPTG (Sigma) was added to a final concentration 1 mM.

Protocol tips

One Shot BL21(DE3) chemically competent E. coli bacteria (Invitrogen) were transformed, according to the manufacturer’s protocol, with 10 ng of each of the 26 pET-BccI plasmids carrying the BRP gene, 25 pET-BccI plasmids with the CAT gene and 22 pET-BccI plasmids carrying the CSFVRP gene. Two colonies from each transformation reaction were used to inoculate equal numbers of 3 ml LB/kanamycin pre-cultures, which were incubated overnight at 37°C and at 250 rpm. The following day, 15 ml cultures (LB/kanamycin) were inoculated with the appropriate volume from their corresponding pre-cultures in order to obtain an optical density (OD)600 value 0.1. The main cultures were incubated up to OD600 = 0.6 and then IPTG (Sigma) was added to a final concentration 1 mM.

Publication protocol

One Shot BL21(DE3) chemically competent E. coli bacteria (Invitrogen) were transformed, according to the manufacturer’s protocol, with 10 ng of each of the 26 pET-BccI plasmids carrying the BRP gene, 25 pET-BccI plasmids with the CAT gene and 22 pET-BccI plasmids carrying the CSFVRP gene. Two colonies from each transformation reaction were used to inoculate equal numbers of 3 ml LB/kanamycin pre-cultures, which were incubated overnight at 37°C and at 250 rpm. The following day, 15 ml cultures (LB/kanamycin) were inoculated with the appropriate volume from their corresponding pre-cultures in order to obtain an optical density (OD)600 value 0.1. The main cultures were incubated up to OD600 = 0.6 and then IPTG (Sigma) was added to a final concentration 1 mM. Four hours later, the cultures were centrifuged at 10,000 g, at 4°C for 5 minutes. The cell pellets were dissolved with 6 volumes of 1× SDS loading buffer and subjected to three instant freeze-thaw cycles from 95°C to -80°C and then finally stored at -18°C. Then 5 μl from each lysate were used for Western blot analysis after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Also an equal volume of PiNK Prestained Protein Marker (Nippon Genetics) was used as a molecular weight indicator. The protein bands were electro-transferred to polyvinyl difluoride membranes (Macherey-Nagel) and, after blocking with 2% bovine serum albumin in phosphate buffered saline (PBS), a 1:2000 dilution of a mouse monoclonal anti-polyHistidine-Peroxidase antibody (Sigma) was used for detection of the expressed 6-Histidine (His) tagged proteins, followed by 3,3′-Diaminobenzidine (DAB) staining (500 μg/ml DAB (Sigma), 2 mM NiCl2 (Sigma) and 0.02% H2O2 (Sigma) in PBS).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Prokaryotic cells - E. coli BRP using 26 pET-BccI from Konstantinos Poulas, Department of Pharmacy, University of Patra.

Manufacturer protocol

Download the product protocol from Konstantinos Poulas, Department of Pharmacy, University of Patra for 26 pET-BccI below.

Videos

Check out videos that might be relevant for performing Protein Expression Prokaryotic cells - E. coli BRP using 26 pET-BccI from Konstantinos Poulas, Department of Pharmacy, University of Patra. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.