Acella™ Chemically Competent Cells
Contents
1. A EdgeBio Acella Chemically Competent Cells Product Cat No Transformations Acella Chemically 36795 12 Competent Cells Tubes Description Acella Chemically Competent Cells are AendA ArecA derivatives of BL21 DE3 cells that have been manufactured using proprietary technology to make the cells highly efficient for DNA uptake thus ultra competent To utilize the cells at their highest efficiency a recommended transformation protocol is included with each kit The complete deletions of the endA and recA genes eliminate plasmid recombination and provide excellent yield and quality plasmid DNA making this strain ideal to combine direct cloning and protein expression By cloning directly in the Acella strain you save at least two days of work normally spent on subcloning and you eliminate the need for additional highly efficient competent cells for cloning procedures Cloning and expression steps can be combined by directly transforming the Acella Chemically Competent Cells with the ligation products using the enclosed protocol Transformed cells should be plated on selective media and incubated at 37 C overnight Since Acella Chemically Competent Cells are fast growing miniprep cultures can be grown for 3 4 hours and plasmids can be analyzed the day after the transformation saving one extra day Positive clones can then be grown directly for protein expression Like all BL21 cells Acella Chemically Compet2. ent Cells lack the Lon and OmpT proteases promoting stability of recombinant proteins Acella Chemically Competent Cells carry a copy of the T7 RNA polymerase and are ideal for expression of T7 promoter driven constructs Acella Chemically Competent Cells are available in single use tubes that provide a simple and reliable method for highly efficient transformations All kits include a test plasmid for quality control purposes Cells are pre dispensed in 50uI aliquots Full processing time including recovery is about one hour 20 minutes Edge BioSystems guarantees transformation efficiencies of Acella Chemically Competent Cells 2 2x10 cfu ug pUC19 Warning This product is intended for research use only It is not to be used for diagnostic purposes in humans or animals Genotype Acella Chemically Competent Cells F ompT hsdSeg rs me gal dcm DE3 AendA ArecA Kit Components 36795 Acella Chemically 12 tubes Competent Cells pUC19 Supercoiled 1 tube DNA 100ng ml Quality Control Each lot is tested to assure high transformation efficiency using 10pg pUC19 supercoiled DNA and the recommended protocol Transformation efficiency will be gt 2x10 cfu ug pUC19 under these conditions Equipment and Materials Not Provided 1 A 42 C water bath 2 14ml round bottom culture tubes 1 per tube of Acella Chemically Competent Cells 3 An orbital shaker capable of 37 C and 320 rpm 4 SOC medium for
3. es or incubate into selective liquid medium For the control transformation with pUC19 supercoiled DNA plate 10 ul on LB ampicillin agar plates and expect gt 20 colonies gt 2 x 10 cfu ug pUC19 Additional Notes a Transformation efficiencies for ligation mixtures will be 10 100 fold lower than for pUC19 supercoiled DNA b Calculation of transformation efficiency cfu on control plate pg of supercoiled pUC19 x 10 pg ug x final dilution cfu ug DNA Note cfu colony forming units Special Note Acella Chemically Competent Cells are based on the T7 expression system This technology was developed at Brookhaven National Laboratory under contract with the U S Department of Energy Consequently U S patents assigned to Brookhaven Science Associates BSA protect this technology These materials are to be used by noncommercial entities for research purposes only Commercial entities require a license from BSA You may refuse these cells by returning the enclosed materials unused To obtain information about licensing please contact the Office of Intellectual Property and Partnerships Brookhaven National Laboratory Building 475D Upton NY 11973 telephone 631 344 7134 or fax 631 344 3729 Warning This product is intended for research use only It is not to be used for diagnostic purposes in humans or animals Page 2 of 2 v 2
4. recovery 20g I tryptone 5g I yeast extract 10mM NaCl 2 5mM KCI 10MM MgClo 10mM MgSO z 20mM glucose MgCl2 MgSO and glucose should be added after autoclaving 5 LB agar plates or liquid media containing the appropriate antibiotic Storage Conditions Acella Chemically Competent Cells should be stored in a 80 C freezer Please note that competent cells are very sensitive to cycles of freezing and thawing and should not be exposed to temperature variations Recommended Protocol for Tubes 1 Immediately after taking the tubes from the 80 C freezer place them on ice for approximately 5 minutes to thaw 2 Pipette the DNA to be transformed to the bottom of the tube and mix by pipetting 50 ul of air to the bottom of the Page 1 of 2 tube Control transformation Dilute pUC19 supercoiled DNA 1 10 with dH20 then add 1 ul of the diluted pUC19 supercoiled DNA to one of the tubes Discard the remaining diluted pUC19 supercoiled DNA after use Note Do not mix by pipetting up and down since that will lower the transformation efficiency 3 Incubate the tubes on ice for 10 minutes 4 Transfer the tubes to a 42 C water bath incubate for 40 seconds and transfer back to ice 5 Incubate the tubes for 2 minutes on ice 6 Transfer the cells into a 14 ml round bottom culture tube filled with 1 ml of pre warmed SOC medium and then shake at 300 rpm at 37 C for 1 hour 7 Plate cells on pre warmed LB agar selective plat
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