STRATAGENE XL1-Blue MRF Supercompetent Cells
Contents
1. autoclaved to a final volume of 100 ml LB Agar per Liter 10 g of NaCl 10 g of tryptone 5 g of yeast extract 20 g of agar Add deionized H30 to a final volume of 1 liter Adjust pH to 7 0 with 5 N NaOH and then autoclave Pour into petri dishes 25 ml 100 mm plate LB Ampicillin Agar per Liter 1 liter of LB agar autoclaved and cooled to 55 C Add 10 ml of 10 mg ml filter sterilized ampicillin Pour into petri dishes 25 ml 100 mm plate Plates for Blue White Color Screening Prepare the LB agar and when adding the antibiotic also add 5 bromo 4 chloro 3 inodlyl B D galactopyranoside X gal to a final concentration of 80 ug ml prepared in dimethylformamide DMF and isopropyl 1 thio B D galactopyranoside IPTG to a final concentration of 20 mM prepared in sterile dH20 Alternatively 100 ul of 10 mM IPTG and 100 ul of 2 X gal may be spread on solidified LB agar plates 30 minutes prior to plating the transformations For consistent color development across the plate pipet the X gal and the IPTG into a 100 1 pool of SOC medium and then spread the mixture across the plate Do not mix the IPTG and the X gal before pipetting them into the pool of SOC medium because these chemicals may precipitate 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 Ag2. white while colonies containing plasmids without inserts will be blue If an insert is suspected to be toxic plate the cells on media without X gal and IPTG Color screening will be eliminated but lower levels of the potentially toxic protein will be expressed in the absence of IPTG Page 2 of 2 Critical Success Factors and Troubleshooting Preparation of Media and Reagents Limited Product Warranty Use of 14 ml BD Falcon polypropylene round bottom tubes It is important that 14 ml BD Falcon polypropylene round bottom tubes BD Biosciences Catalog 352059 are used for the transformation protocol since other tubes may be degraded by B mercaptoethanol In addition the duration of the heat pulse has been optimized using these tubes Aliquoting Cells Keep the cells on ice at all times during aliquoting It is essential that the polypropylene tubes are placed on ice before the cells are thawed and that the cells are aliquoted directly into pre chilled tubes It is also important to use the volume of cells indicated in step 2 of the Transformation Protocol Decreasing the volume will reduce efficiency Use of B Mercaptoethanol B ME B ME has been shown to increase transformation efficiency The B ME mixture provided is diluted and ready to use A fresh 1 10 dilution from a 14 2 M stock may be used however Stratagene cannot guarantee results with B ME from other sources Quantity and Volume of DNA The greatest efficiency is obtained from
3. 6 Incubate the tubes on ice for 30 minutes 7 Heat pulse the tubes in a 42 C water bath for 45 seconds The duration of the heat pulse is critical 8 Incubate the tubes on ice for 2 minutes 9 Add 0 9 ml of preheated 42 C SOC medium and incubate the tubes at 37 C for 1 hour with shaking at 225 250 rpm 10 Plate lt 200 ul of the transformation mixture on LB agar plates containing the appropriate antibiotic and containing IPTG and X gal if color screening is desired For the pUC18 control transformation plate 2 5 ul of the transformation mixture on LB ampicillin agar plates 11 Incubate the plates at 37 C overnight If performing blue white color screening incubate the plates at 37 C for at least 17 hours to allow color development color can be enhanced by subsequent incubation of the plates for 2 hours at 4 C 12 For the pUC18 control expect 250 colonies 21 x 10 cfu ug pUC18 DNA For the experimental DNA the number of colonies will vary according to the size and form of the transforming DNA with larger and non supercoiled DNA producing fewer colonies Blue white color screening for recombinant plasmids is available when transforming this host strain containing the lac ZAM15 gene on the F episome with a plasmid that provides a complementation e g the Stratagene pBluescript II vector When lacZ expression is induced by IPTG in the presence of the chromogenic substrate X gal colonies containing plasmids with inserts will be
4. AM15 Tn10 Tet Genes listed signify mutant alleles Genes on the F episome however are wild type unless indicated otherwise The XL1 Blue MRF Minus Restriction strain is a restriction minus McrA McrCB McrF Mrr HsdR_ derivative of Stratagene s XL1 Blue strain XL1 Blue MRF cells are deficient in all known restriction systems A mcrA 183 A mcrCB hsdSMR mrr 173 and are endonuclease endA and recombination recA deficient The hsdR mutation prevents the cleavage of cloned DNA by the EcoK endonuclease system and the recA mutation helps ensure insert stability The endA mutation greatly improves the quality of miniprep DNA The lac ZAM15 gene on the F episome allows blue white screening for recombinant plasmids XL1 Blue MRF cells are resistant to tetracycline Pre chill two 14 ml BD Falcon polypropylene round bottom tubes on ice One tube is for the experimental transformation and one tube is for the pUC18 control Preheat SOC medium to 42 C Thaw the cells on ice When thawed gently mix and aliquot 100 ul of cells into each of the two pre chilled tubes Add 1 7 ul of the B mercaptoethanol provided with this kit to each aliquot of cells Swirl the tubes gently Incubate the cells on ice for 10 minutes swirling gently every 2 minutes Add 0 1 50 ng of the experimental DNA to one aliquot of cells and add 1 ul of the pUC18 control DNA to the other aliquot Swirl the tubes gently nA kwh
5. Page 1 of 2 Catalog Number Product Name Materials Provided Certified By Quality Controlled By Shipping Conditions Storage Conditions Guaranteed Efficiency Test Conditions Genotype and Background Antibiotic Resistance Transformation Protocol Blue White Color Screening STRATAGENE An Agilent Technologies Division Made in USA 200230 XL1 Blue MRF Supercompetent Cells XL1 Blue MRF supercompetent cells yellow tubes 5 x 200 ul pUC18 control plasmid 0 1 ng ul in TE buffer 10 pl B Mercaptoethanol 1 42 M 25 ul Todd Parsons Tricia Molina Shipped on dry ice Competent cells must be placed immediately at the bottom of a 80 C freezer directly from the dry ice shipping container Do not store the cells in liquid nitrogen Competent cells are sensitive to even small variations in temperature Transferring tubes from one freezer to another may result in a loss of efficiency gt 1 0 x 10 cfu g pUC18 DNA Transformations are performed both with and without plasmid DNA using 100 ul aliquots of cells and 100 pg of pUC18 control DNA following the protocol outlined below Following transformation 2 5 11 samples of the culture are plated in duplicate on LB agar plates with 100 ug ml ampicillin The plates are incubated at 37 C overnight and the efficiency is calculated based on the average number of colonies per plate A mcrA 183 A mcrCB hsdSMR mrr 173 endA1 supE44 thi 1 recAl gyrA96 relA1 lac F proAB lacI Z
6. ilent Agilent 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 For in vitro use only This certificate is a declaration of analysis at the time of manufacture
7. the transformation of 1 ul of 0 1 ng ul supercoiled pUC18 DNA per 100 ul of cells A greater number of colonies may be obtained by transforming up to 50 ng DNA although the resulting efficiency cfu ug may be lower The volume of the DNA solution added to the reaction may be increased to up to 10 of the reaction volume but the transformation efficiency may be reduced Heat Pulse Duration and Temperature Optimal transformation efficiency is observed when cells are heat pulsed at 42 C for 45 50 seconds Efficiency decreases sharply when cells are heat pulsed for lt 45 seconds or for gt 60 seconds Do not exceed 42 C Plating the Transformation Mixture If plating lt 100 ul of cells pipet the cells into a 200 11 pool of SOC medium and then spread the mixture with a sterile spreader If plating gt 100 ul the cells can be spread on the plates directly Tilt and tap the spreader to remove the last drop of cells If desired cells may be concentrated prior to plating by centrifugation at 1000 rpm for 10 minutes followed by resuspension in 200 ul of SOC medium SOB Medium per Liter 20 0 g of tryptone 5 0 g of yeast extract 0 5 g of NaCl Add deionized H30 to a final volume of 1 liter and then autoclave Add 10 ml of filter sterilized 1 M MgCl and 10 ml of filter sterilized 1 M MgSO prior to use SOC Medium per 100 ml Prepare immediately before use 2 ml of filter sterilized 20 w v glucose or 1 ml of filter sterilized 2 M glucose SOB medium
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