STRATAGENE BacterioMatch II Electrocompetent Reporter Cells
Contents
1. QUALITY CONTROL TESTING Transformations are performed both with and without pUC18 DNA using 40 ul aliquots of cells and 1 ul of pUC18 plasmid 10 pg ul following the protocol outlined above Following transformation 5 ul samples of the culture are plated in duplicate on LB agar plates with 100 ug ml of ampicillin The plates are incubated at 37 C overnight and the efficiency is calculated based on the average number of colonies per plate Full activity is guaranteed through two years Lot Efficiency gt 1 x 10 cfu ug pUCI8 DNA Certified By Quality Controlled By NOTICE TO PURCHASER USS Patent No 5 925 523 covering the BacterioMatch two hybrid system is licensed exclusively by Stratagene Research use of the BacterioMatch two hybrid system by commercial entities requires a license from Stratagene For license information please contact Director of Business Development at 858 535 5400 ext 13043 LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product No other warranties of any kind express or implied including without limitation implied warranties of merchantability or fitness for a particular purpose are provided by Stratagene Stratagene shall have no liability for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product ENDNOTES BacterioMatch is a registered trademark of Stratagene in th2. BacterioMatch II Electrocompetent Reporter Cells Catalog 2001 95 UNA OU OU DODN UNLU TOLOA QUN UA OAU A Storage Store the cells immediately at the bottom of a 80 C freezer Do not store the cells in liquid nitrogen Store the control plasmid DNA at 80 C INTRODUCTION The BacterioMatch II two hybrid system reporter strain is a histidine auxotrophic mutant derived from XL1 Blue MRF This strain harbors the genetic elements necessary to report the interaction between recombinant bait and target proteins expressed from BacterioMatch II two hybrid system plasmids Episomally expressed LaqI represses synthesis of the bait and target until induction Positive protein protein interactions are indicated by the expression of a reporter gene cassette that includes the H S3 gene conferring the ability to grow in the presence of the compound 3 AT and aadA gene conferring streptomycin resistance BacterioMatch II electrocompetent reporter cells catalog 200195 are optimized for high efficiency large scale cotransformation reactions performed during BacterioMatch II two hybrid library screening Performing library cotransformation by electroporation greatly increases the transformation efficiency decreasing the number of cotransformation reactions required for library coverage For the small scale cotransformation of purified plasmids performed during BacterioMatch II two hybrid system validation steps Stratagene offers the chemically compe
3. ant to use pUC18 plasmid DNA Do not substitute with pBT pTRG or other plasmids For a protocol for cotransformation of reporter strain electrocompetent cells with Bait and Target plasmids see the BacterioMatch II Two Hybrid System Library Construction Kit with Electrocompetent Cells Instruction Manual available online at http www stratagene com manuals index asp Search for catalog 200414 1 Pre chill a sterile electroporation cuvette 0 1 cm gap and a sterile 1 5 ml microcentrifuge tube thoroughly on ice Preheat sterile SOC medium to 37 C 2 Set the electroporator to a voltage setting of 1500 V 15 kV cm field strength Set the resistance to 400 Q and the capacitance to 25 uF 3 Thaw the BacterioMatch II electrocompetent reporter cells on ice After mixing the cells gently aliquot 40 ul of cells into the pre chilled microcentrifuge tube Keep the tube on ice throughout the procedure Dilute the pUC18 control DNA 1 10 with sterile dH O Add 1 ul of the diluted pUC18 DNA to the cells with gentle mixing Transfer the cell DNA mixture to a chilled electroporation cuvette tapping the cuvette until the mixture settles evenly to the bottom Slide the cuvette into the electroporation chamber until the cuvette sits flush against the electrical contacts Pulse the sample once then quickly remove the cuvette Immediately add 960 ul of SOC medium held at 37 C to resuspend the cells Transfer the cells to a sterile 14 ml BD Falcon
4. e United States Manual Part 200195 11 Revision 074002 Copyright 2004 by Stratagene
5. ential that the microcentrifuge tubes that the cells will be aliquoted into are placed on ice before the cells are thawed and that the cells are aliquoted directly into the pre chilled tubes Cuvette Gap Width Use a cuvette with a 0 1 cm gap to maximize the transformation efficiency and to minimize the possibility of arcing A cuvette with a 0 2 cm gap is not recommended because the transformation efficiency is lower and the possibility of arcing is higher lonic Strength of DNA Solution The sample DNA to be transformed by electroporation must be in a low ionic strength buffer such as TE buffer or water DNA samples containing too much salt will cause arcing at high voltage possibly damaging both the sample and the electroporator Plating Media For the efficiency determination assay described in the protocol on the reverse page it is important to plate the cells transformed with the pUC18 plasmid on LB ampicillin medium For plating after cotransformation of the bait plasmid carrying Cam and a target plasmid library carrying Tet during two hybrid interaction screens plating should be performed according to the instruction manual provided with the BacterioMatch II Two Hybrid System Library Construction Kit with Electrocompetent Cells Catalog 200414 available online at http www stratagene com manuals index asp ELECTROPORATION PROTOCOL FOR DETERMINATION OF TRANSFORMATION EFFICIENCY Note For the efficiency determination it is import
6. polypropylene round bottom tube BD Biosciences Catalog 352059 Incubate the tube at 37 C for 60 minutes with shaking at 225 rpm 9 Following the incubation period plate 5 ul of the transformation mixture onto each of two LB ampicillin agar plates To accommodate this small plating volume first place a 200 11 pool of SOC medium on the plate Pipet 5 ul of the cells into the pool and then spread the mixture with a sterile spreader 10 Incubate the plates overnight at 37 C inverted 11 Count the number of colonies on each plate Expect an average of 250 cfu indicating an efficiency of 21 x 10 cfu ug pUC18 DNA oOo NDAU A PREPARATION OF MEDIA AND REAGENTS SOB Medium per Liter SOC Medium per 100 ml T ie eee i Note This medium should be prepared immediately before 0 5 g of NaCl satan ts a Add deionized H O to a final volume of 1 liter 2 ml of filter sterilized 20 w v glucose or 1 ml of filter sterilized Autoclave 2M glucose Add 10 ml of filter sterilized 1 M MgCl and 10 ml of filter sterilized SOB medium autoclaved to a final volume of 100 ml 1 M MgSO prior to use LB Agar per Liter LB Ampicillin Agar per Liter 10 g of NaCl Prepare 1 liter of LB agar 10 g of tryptone Autoclave 5 g of yeast extract Cool to 55 C 20 g of agar Add 10 ml of 10 mg ml filter sterilized ampicillin Adjust pH to 7 0 with 5 N NaOH Pour into petri dishes 25 ml 100 mm plate Add deionized H O to a final volume of 1 liter Autoclave
7. tent BacterioMatch II validation reporter competent cells catalog 200192 Stratagene also offers chemically competent cells for library screening BacterioMatch II screening reporter competent cells catalog 200190 MATERIALS PROVIDED Transformation Efficiency Materials Provided Quantity cfu ug of pUC18 DNA BacterioMatch II electrocompetent reporter cells 5 x 100 ul 1x 10 pUC18 plasmid DNA 0 1 ng ul in TE buffer 10 ul a Stratagene guarantees this efficiency when the cells are used according to the protocol in this instruction manual ADDITIONAL MATERIALS REQUIRED Sterile electroporation cuvettes 0 1 cm gap width Sterile microcentrifuge tubes 14 ml BD Falcon polypropylene round bottom tubes BD Biosciences Catalog 352059 GENOTYPE A mcrA 183 A mcrCB hsdSMR mrr 173 endAl hisB supE44 thi 1 recAl gyrA96 relA1 lac F laql HIS3 aadA Kan BacterioMatch II electrocompetent cells are kanamycin resistant TRANSFORMATION GUIDELINES FOR BACTERIOMATCH II TWO HYBRID SYSTEM REPORTER STRAIN COMPETENT CELLS Storage Conditions Electrocompetent cells are sensitive to even small variations in temperature and must be stored at the bottom of a 80 C freezer Transferring tubes from one freezer to another may result in a loss of efficiency Competent cells should be placed at 80 C directly from the dry ice shipping container Aliquoting Cells Keep the cells on ice at all times during aliquoting It is ess
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